Therapeutic nuclease compositions and methods

ABSTRACT

Hybrid nuclease molecules and methods for treating an immune-related disease or disorder in a mammal, and a pharmaceutical composition for treating an immune-related disease in a mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/257,458, filed Nov. 2, 2009, and U.S. Provisional Application No.61/370,752, filed Aug. 4, 2010, the entire disclosures of which arehereby incorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with support from the National Institutes ofHealth (Grants AI44257, NS065933, and AR048796), the Alliance for LupusResearch, and the Washington State Life Science Discovery Fund(2087750). The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named XXXXXPCT_sequencelisting.txt, created on Month X,20XX, with a size of X bytes. The sequence listing is incorporated byreference.

BACKGROUND

Excessive release of (ribo)nucleoprotein particles from dead and dyingcells can cause lupus pathology by two mechanisms: (i) Deposition or insitu formation of chromatin/anti-chromatin complexes causes nephritisand leads to loss of renal function; and (ii) nucleoproteins activateinnate immunity through toll-like receptor (TLR) 7, 8, and 9 as well asTLR-independent pathway(s). Release of nucleoproteins can serve as apotent antigen for autoantibodies in SLE, providing amplification of Bcell and DC activation through co-engagement of antigen receptors andTLRs. Thus, there exists a need for a means to remove inciting antigensand/or attenuate immune stimulation, immune amplification, and immunecomplex mediated disease in subjects in need thereof.

SUMMARY

Disclosed herein is a hybrid nuclease molecule comprising a firstnuclease domain and an Fc domain, wherein the first nuclease domain isoperatively coupled to the Fc domain. In some embodiments, the hybridnuclease molecule further includes a first linker domain, and the firstnuclease domain is operatively coupled to the Fc domain by the firstlinker domain.

In some embodiments, a hybrid nuclease molecule is a polypeptide,wherein the amino acid sequence of the first nuclease domain comprises ahuman, wild-type RNase amino acid sequence, wherein the first linkerdomain is (Gly4Ser)n, where n is 0, 1, 2, 3, 4 or 5, wherein the aminoacid sequence of the Fc domain comprises a human, wild-type IgG1 Fcdomain amino acid sequence, and wherein the first linker domain iscoupled to the C-terminus of the first nuclease domain and theN-terminus of the Fc domain. In some embodiments, a hybrid nucleasemolecule is a polypeptide comprising or consisting of a sequence shownin Table 2. In some embodiments, a hybrid nuclease molecule is apolypeptide comprising SEQ ID NO:149. In some embodiments, a hybridnuclease molecule is a polypeptide comprising SEQ ID NO:145. In someembodiments, a hybrid nuclease molecule is a polypeptide comprising SEQID NO:161. In some embodiments, a hybrid nuclease molecule is apolypeptide comprising SEQ ID NO:162. In some embodiments, a hybridnuclease molecule is a polypeptide comprising SEQ ID NO:163.

In some embodiments, a hybrid nuclease molecule comprises wild-type,human DNase1 linked to wild-type, human IgG1. In some embodiments, ahybrid nuclease molecule comprises human DNase1 G105R A114F linked to awild-type, human IgG1 Fc domain by a (gly4ser)n linker domain where n=0,1, 2, 3, 4, or 5. In some embodiments, a hybrid nuclease moleculecomprises wild-type, human RNase1 linked to wild-type, human IgG1 linkedto wild-type, human DNase1. In some embodiments, a hybrid nucleasemolecule comprises wild-type, human RNase1 linked to wild-type, humanIgG1 linked to human DNase1 G105R A114F. In some embodiments, a hybridnuclease molecule is a polypeptide, wherein the amino acid sequence ofthe first nuclease domain comprises a RNase amino acid sequence, whereinthe first linker domain is between 5 and 32 amino acids in length,wherein the amino acid sequence of the Fc domain comprises a human, Fcdomain amino acid sequence, and wherein the first linker domain iscoupled to the C-terminus of the first nuclease domain and theN-terminus of the Fc domain. In some embodiments, the linker domainincludes (gly4ser)5 and restriction sites BglII, AgeI, and XhoI. In someembodiments, a hybrid nuclease molecule is a polypeptide, wherein theamino acid sequence of the first nuclease domain comprises a human RNaseamino acid sequence, wherein the first linker domain is a NLG peptidebetween 5 and 32 amino acids in length, wherein the amino acid sequenceof the Fc domain comprises a human, wild-type Fc domain amino acidsequence, and wherein the first linker domain is coupled to theC-terminus of the first nuclease domain and the N-terminus of the Fcdomain.

In some embodiments, the Fc domain binds to an Fc receptor on a humancell. In some embodiments, the serum half-life of the molecule issignificantly longer than the serum half-life of the first nucleasedomain alone. In some embodiments, the nuclease activity of the firstnuclease domain of the molecule is the same or greater than the nucleasedomain alone. In some embodiments, administration of the molecule to amouse increases the survival rate of the mouse as measured by a mouseLupus model assay.

In some embodiments, a hybrid nuclease molecule includes a leadersequence. In some embodiments, the leader sequence is human VK3LPpeptide from the human kappa light chain family, and the leader sequenceis coupled to the N-terminus of the first nuclease domain.

In some embodiments, the molecule is a polypeptide. In some embodiments,the molecule is a polynucleotide.

In some embodiments, the first nuclease domain comprises an RNase. Insome embodiments, the RNase is a human RNase. In some embodiments, theRNase is a polypeptide comprising an amino acid sequence at least 90%similar to an RNase amino acid sequence set forth in Table 2. In someembodiments, the RNase is a human RNase A family member. In someembodiments, the RNase is a human pancreatic RNase1.

In some embodiments, the first nuclease domain comprises a DNase. Insome embodiments, the DNase is a human DNase. In some embodiments, theDNase is a polypeptide comprising an amino acid sequence at least 90%similar to a DNase amino acid sequence set forth in Table 2. In someembodiments, the DNase is selected from the group consisting of humanDNase I, TREX1, and human DNase 1L3.

In some embodiments, the Fc domain is a human Fc domain. In someembodiments, the Fc domain is a wild-type Fc domain. In someembodiments, the Fc domain is a mutant Fc domain. In some embodiments,the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fcdomain is a polypeptide comprising an amino acid sequence at least 90%similar to an Fc domain amino acid sequence set forth in Table 2.

In some embodiments, the first linker domain has a length of about 1 toabout 50 amino acids. In some embodiments, the first linker domain has alength of about 5 to about 31 amino acids. In some embodiments, thefirst linker domain has a length of about 15 to about 25 amino acids. Insome embodiments, the first linker domain has a length of about 20 toabout 32 amino acids. In some embodiments, the first linker domain has alength of about 20 amino acids. In some embodiments, the first linkerdomain has a length of about 25 amino acids. In some embodiments, thefirst linker domain has a length of about 18 amino acids. In someembodiments, the first linker domain comprises a gly/ser peptide. Insome embodiments, the gly/ser peptide is of the formula (Gly₄Ser)n,wherein n is a positive integer selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the gly/ser peptideincludes (Gly₄Ser)3. In some embodiments, the gly/ser peptide includes(Gly₄Ser)4. In some embodiments, the gly/ser peptide includes(Gly₄Ser)5. In some embodiments, the first linker domain includes atleast one restriction site. In some embodiments, the first linker domainincludes about 12 or greater nucleotides including at least onerestriction site. In some embodiments, the first linker domain includestwo or more restriction sites. In some embodiments, the first linkerdomain includes a plurality of restriction sites. In some embodiments,the first linker domain comprises an NLG peptide. In some embodiments,the first linker domain comprises an N-linked glycosylation site.

In some embodiments, the first nuclease domain is linked to theN-terminus of the Fc domain. In some embodiments, the first nucleasedomain is linked to the C-terminus of the Fc domain.

In some embodiments, the hybrid nuclease molecule further includes asecond nuclease domain. In some embodiments, the first and secondnuclease domains are distinct nuclease domains. In some embodiments, thefirst and second nuclease domains are the same nuclease domains. In someembodiments, the second nuclease domain is linked to the C-terminus ofthe Fc domain. In some embodiments, the second nuclease domain is linkedto the N-terminus of the Fc domain. In some embodiments, the secondnuclease domain is linked to the C-terminus of the first nucleasedomain. In some embodiments, the second nuclease domain is linked to theN-terminus of the first nuclease domain.

Also disclosed herein is a dimeric polypeptide comprising a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises a first nuclease domain, and an Fc domain, wherein the firstnuclease domain is operatively coupled to the Fc domain. In someembodiments, the second polypeptide is a second hybrid nuclease moleculecomprising a second nuclease domain, and a second Fc domain, wherein thesecond nuclease domain is operatively coupled to the second Fc domain.

Also disclosed herein is a pharmaceutical composition comprising atleast one hybrid nuclease molecule and/or at least one dimericpolypeptide as described herein, and a pharmaceutically acceptableexcipient.

Also disclosed herein is a nucleic acid molecule encoding a hybridnuclease molecule disclosed herein. Also disclosed herein is arecombinant expression vector comprising a nucleic acid moleculedisclosed herein. Also disclosed herein is a host cell transformed witha recombinant expression vector disclosed herein.

Also disclosed herein is a method of making a hybrid nuclease disclosedherein, comprising: providing a host cell comprising a nucleic acidsequence that encodes the hybrid nuclease molecule; and maintaining thehost cell under conditions in which the hybrid nuclease molecule isexpressed.

Also disclosed herein is a method for treating or preventing a conditionassociated with an abnormal immune response, comprising administering toa patient in need thereof an effective amount of an isolated hybridnuclease molecule disclosed herein. In some embodiments, the conditionis an autoimmune disease. In some embodiments, the autoimmune disease isselected from the group consisting of insulin-dependent diabetesmellitus, multiple sclerosis, experimental autoimmune encephalomyelitis,rheumatoid arthritis, experimental autoimmune arthritis, myastheniagravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto'sthyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia,autoimmune atrophic gastritis, Addison's disease, premature menopause,male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis,autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliarycirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis,ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener'sgranulomatosis, polymyositis, dermatomyositis, discoid LE, systemiclupus erythematosus (SLE), and connective tissue disease. In someembodiments, the autoimmune disease is SLE.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows the nucleotide and amino acid sequence of the mRNase-mIgG2awith mutations at P238S, K322S, and P331 S. This sequence is listed inthe sequence listing as huVK3LP+mrib1+mIgG2A-C-2S (SEQ ID NO:114).

FIG. 2 shows a schematic diagram of some embodiments of hybrid nucleasemolecules described herein.

FIG. 3 shows the SDS-PAGE gel analysis of mRNase-mIgG2a-c under bothreducing and non-reducing conditions.

FIG. 4 shows gel immunoprecipitation analysis of mRNasemIg2a-c.

FIG. 5 shows an anti-RNA antibody ELISA titer before and afterintravenous injection of RNase-Ig hybrid nuclease molecule from mouse410. The data show that injection of RNase-Ig caused a reduction intiter of anti-RNA antibody that persisted for over 3 weeks.

FIG. 6 shows that RNase-Ig addition abolished the induction ofinterferon-α from human peripheral blood mononuclear cells stimulatedusing immune complexes formed with serum from an SLE patient (J11) plusnuclear extract (NE). Titer of anti-RNA antibody was reduced afterinjection of RNase-Ig.

FIG. 7 shows that RNase-Ig addition abolished the induction ofinterferon-α from human peripheral blood mononuclear cells stimulatedusing immune complexes formed with serum from an SLE patient (J11) plusnuclear extract.

FIG. 8 shows single radial enzyme diffusion (SRED) analysis of serumfrom two RNase transgenic (Tg) mice compared to a normal B6 mouse.

FIG. 9 shows the concentration of RNaseA in Tg and double Tg (DTg) micemeasured by ELISA. Each dot represents the concentration measured in anindividual mouse.

FIG. 10 shows survival of TLR7.1 Tg versus TLR7.1xRNaseA DTg mice

FIG. 11 shows quantitative PCR of IRGs in spleens of Tg versus DTg mice.

FIG. 12 shows a prototype structure for creating different embodimentsof hybrid nuclease molecules.

FIG. 13 shows the enzyme kinetics for hRNase1-G88D-hIgG1SCCH-P238S-K322S-P331S hybrid nuclease molecules as measured using RNaseAlert Substrate™.

FIG. 14 shows the binding of hRNase1-WT-hIgG1-WT to human monocytic celllines U937 and THP1. The peak on the left in both plots is control andthe peak on the right in both plots is hRNase1-WT-hIgG1-WT.

FIG. 15 shows the blocking activity of human IVIg for binding to U937and THP-1 cells by hRNase1-WT-hIgG1-WT.

FIG. 16 shows the results of a DNA digestion assay by Trex1-(g4s)n-mIgGalternative forms.

FIG. 17 shows the results of a Western Blot for trex1-(Gly4S)4-Ig andtrex1-(Gly4S)5-Ig culture supernatants from COS-7 transienttransfections.

FIG. 18 shows DNA digestion patterns by different stably transfected CHODG44 clones designated as 2A3, 3A5, and 8H8, expressingDNAse1L3-mIgG2a-c hybrid nuclease molecules.

FIG. 19 shows DNA digestion patterns of decreasing amounts ofDNase1L3-Ig hybrid nuclease molecules after various incubation timeswith and without heparin as an enzyme inhibitor.

FIG. 20 shows a Western blot of immuneprecipitated fusion proteins fromtransiently transfected COS cells expressing different embodiments ofhRNase1-Ig-hDNase1 or hDNase1-Ig hybrid nuclease molecules.

FIG. 21 shows SRED analysis to assess the presence of RNase activity inthe COS supernatants expressing different embodiments ofhRNase1-Ig-hDNase1 or hDNase1-Ig hybrid nuclease molecules.

FIG. 22 shows a composite figure displaying results of DNase nucleaseactivity assays performed on COS supernatants from transfected cells.The description of the numbering (e.g., 090210-8 and 091210-8) from FIG.21 applies to this figure as well.

FIG. 23 shows enzyme kinetics assayed using the Rnase Alert Substrate(Ambion/IDT) and fluorescence quantified with a Spectramax M2 microplateReader. Data was analyzed using Softmax Pro software (MolecularDevices). Reaction rates at different substrate concentrations weremeasured and the data shown as a Lineweaver-Burk plot. The apparent Km,corrected for volume is 280 nM.

FIG. 24 shows the levels of anti-RNA antibodies in mouse sera from H564and H564-RNaseA double transgenic mice at successive intervals as thetransgenic mice aged.

DETAILED DESCRIPTION

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant specification shall control.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions,” can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al.,1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginineand leucine, modifications at the second base can also be conservative.The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

Polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, the term “hybrid nuclease molecule” refers topolynucleotides or polypeptides that comprise at least one nucleasedomain and at least one Fc domain. Hybrid nuclease molecules are alsoreferred to as fusion protein(s) and fusion gene(s). For example, in oneembodiment, a hybrid nuclease molecule can be a polypeptide comprisingat least one Fc domain linked to a nuclease domain such as DNase and/orRNase. As another example, a hybrid nuclease molecule can include anRNase nuclease domain, a linker domain, and an Fc domain. SEQ ID NO:161is an example of a hybrid nuclease molecule. Other examples aredescribed in more detail below. In one embodiment a hybrid nucleasemolecule of the invention can include additional modifications. Inanother embodiment, a hybrid nuclease molecule may be modified to add afunctional moiety (e.g., PEG, a drug, or a label).

In certain aspects, the hybrid nuclease molecules of the invention canemploy one or more “linker domains,” such as polypeptide linkers. Asused herein, the term “linker domain” refers to a sequence whichconnects two or more domains in a linear sequence. As used herein, theterm “polypeptide linker” refers to a peptide or polypeptide sequence(e.g., a synthetic peptide or polypeptide sequence) which connects twoor more domains in a linear amino acid sequence of a polypeptide chain.For example, polypeptide linkers may be used to connect a nucleasedomain to an Fc domain. Preferably, such polypeptide linkers can provideflexibility to the polypeptide molecule. In certain embodiments thepolypeptide linker is used to connect (e.g., genetically fuse) one ormore Fc domains and/or one or more nuclease domains. A hybrid nucleasemolecule of the invention may comprise more than one linker domain orpeptide linker.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser polypeptide linker comprises the amino acid sequenceSer(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2. Inanother embodiment, n=3, i.e., Ser(Gly₄Ser)3. In another embodiment,n=4, i.e., Ser(Gly₄Ser)4. In another embodiment, n=5. In yet anotherembodiment, n=6. In another embodiment, n=7. In yet another embodiment,n=8. In another embodiment, n=9. In yet another embodiment, n=10.Another exemplary gly/ser polypeptide linker comprises the amino acidsequence Ser(Gly₄Ser)n. In one embodiment, n=1. In one embodiment, n=2.In a preferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6.

As used herein, the terms “linked,” “fused”, or “fusion”, are usedinterchangeably. These terms refer to the joining together of two moreelements or components or domains, by whatever means including chemicalconjugation or recombinant means. Methods of chemical conjugation (e.g.,using heterobifunctional crosslinking agents) are known in the art.

As used herein, the term “Fc region” shall be defined as the portion ofa native immunoglobulin formed by the respective Fc domains (or Fcmoieties) of its two heavy chains.

As used herein, the term “Fc domain” refers to a portion of a singleimmunoglobulin (Ig) heavy chain. As such, Fc domain can also be referredto as “Ig” or “IgG.” In some embodiments, an Fc domain begins in thehinge region just upstream of the papain cleavage site and ending at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain. In certainembodiments, an Fc domain comprises at least one of: a hinge (e.g.,upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3domain, a CH4 domain, or a variant, portion, or fragment thereof. Inother embodiments, an Fc domain comprises a complete Fc domain (i.e., ahinge domain, a CH2 domain, and a CH3 domain). In one embodiment, an Fcdomain comprises a hinge domain (or portion thereof) fused to a CH3domain (or portion thereof). In another embodiment, an Fc domaincomprises a CH2 domain (or portion thereof) fused to a CH3 domain (orportion thereof). In another embodiment, an Fc domain consists of a CH3domain or portion thereof. In another embodiment, an Fc domain consistsof a hinge domain (or portion thereof) and a CH3 domain (or portionthereof). In another embodiment, an Fc domain consists of a CH2 domain(or portion thereof) and a CH3 domain. In another embodiment, an Fcdomain consists of a hinge domain (or portion thereof) and a CH2 domain(or portion thereof). In one embodiment, an Fc domain lacks at least aportion of a CH2 domain (e.g., all or part of a CH2 domain). In oneembodiment, an Fc domain of the invention comprises at least the portionof an Fc molecule known in the art to be required for FcRn binding. Inanother embodiment, an Fc domain of the invention comprises at least theportion of an Fc molecule known in the art to be required for FcγRbinding. In one embodiment, an Fc domain of the invention comprises atleast the portion of an Fc molecule known in the art to be required forProtein A binding. In one embodiment, an Fc domain of the inventioncomprises at least the portion of an Fc molecule known in the art to berequired for protein G binding. An Fc domain herein generally refers toa polypeptide comprising all or part of the Fc domain of animmunoglobulin heavy-chain. This includes, but is not limited to,polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domainsas well as fragments of such peptides comprising only, e.g., the hinge,CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulinof any species and/or any subtype, including, but not limited to, ahuman IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fcdomain encompasses native Fc and Fc variant molecules. As with Fcvariants and native Fc's, the term Fc domain includes molecules inmonomeric or multimeric form, whether digested from whole antibody orproduced by other means.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain exemplary embodiments, the Fc domainretains an effector function (e.g., FcγR binding).

The Fc domains of a polypeptide of the invention may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence.

Polypeptides derived from another peptide may have one or more mutationsrelative to the starting polypeptide, e.g., one or more amino acidresidues which have been substituted with another amino acid residue orwhich has one or more amino acid residue insertions or deletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants necessarily have less than 100% sequenceidentity or similarity with the starting hybrid nuclease molecules. In apreferred embodiment, the variant will have an amino acid sequence fromabout 75% to less than 100% amino acid sequence identity or similaritywith the amino acid sequence of the starting polypeptide, morepreferably from about 80% to less than 100%, more preferably from about85% to less than 100%, more preferably from about 90% to less than 100%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferablyfrom about 95% to less than 100%, e.g., over the length of the variantmolecule.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e. same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide of the invention consists of, consistsessentially of, or comprises an amino acid sequence selected from Table2 and functionally active variants thereof. In an embodiment, apolypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence set forth in Table 2. Inan embodiment, a polypeptide includes a contiguous amino acid sequenceat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguousamino acid sequence set forth in Table 2. In an embodiment, apolypeptide includes an amino acid sequence having at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200,300, 400, or 500 (or any integer within these numbers) contiguous aminoacids of an amino acid sequence set forth in Table 2.

In an embodiment, the peptides of the invention are encoded by anucleotide sequence. Nucleotide sequences of the invention can be usefulfor a number of applications, including: cloning, gene therapy, proteinexpression and purification, mutation introduction, DNA vaccination of ahost in need thereof, antibody generation for, e.g., passiveimmunization, PCR, primer and probe generation, siRNA design andgeneration (see, e.g., the Dharmacon siDesign website), and the like. Inan embodiment, the nucleotide sequence of the invention comprises,consists of, or consists essentially of, a nucleotide sequence selectedfrom Table 2. In an embodiment, a nucleotide sequence includes anucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a nucleotide sequence set forth in Table 2. In an embodiment, anucleotide sequence includes a contiguous nucleotide sequence at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotidesequence set forth in Table 2. In an embodiment, a nucleotide sequenceincludes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous nucleotides of anucleotide sequence set forth in Table 2.

Preferred hybrid nuclease molecules of the invention comprise a sequence(e.g., at least one Fc domain) derived from a human immunoglobulinsequence. However, sequences may comprise one or more sequences fromanother mammalian species. For example, a primate Fc domain or nucleasedomain may be included in the subject sequence. Alternatively, one ormore murine amino acids may be present in a polypeptide. In someembodiments, polypeptide sequences of the invention are not immunogenicand/or have reduced immunogenicity.

It will also be understood by one of ordinary skill in the art that thehybrid nuclease molecules of the invention may be altered such that theyvary in sequence from the naturally occurring or native sequences fromwhich they were derived, while retaining the desirable activity of thenative sequences. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at “non-essential”amino acid residues may be made. An isolated nucleic acid moleculeencoding a non-natural variant of a hybrid nuclease molecule derivedfrom an immunoglobulin (e.g., an Fc domain) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

The peptide hybrid nuclease molecules of the invention may compriseconservative amino acid substitutions at one or more amino acidresidues, e.g., at essential or non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a binding polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members. Alternatively, in another embodiment, mutations may beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be incorporatedinto binding polypeptides of the invention and screened for theirability to bind to the desired target.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an autoimmune disease state(e.g., SLE), including prophylaxis, lessening in the severity orprogression, remission, or cure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

Hybrid Nuclease Molecules

In some embodiments, a composition of the invention includes a hybridnuclease molecule. In some embodiments, a hybrid nuclease moleculeincludes a nuclease domain operatively linked to an Fc domain. In someembodiments, a hybrid nuclease molecule includes a nuclease domainlinked to an Fc domain. In some embodiments the hybrid nuclease moleculeis a nuclease protein. In some embodiments, the hybrid nuclease moleculeis a nuclease polynucleotide.

In some embodiments, the nuclease domain is linked to the Fc domain viaa linker domain. In some embodiments, the linker domain is a linkerpeptide. In some embodiments, the linker domain is a linker nucleotide.In some embodiments, the hybrid nuclease molecule includes a leadermolecule, e.g., a leader peptide. In some embodiments, the leadermolecule is a leader peptide positioned at the N-terminus of thenuclease domain. In some embodiments, the hybrid nuclease molecule willinclude a stop codon. In some embodiments, the stop codon will be at theC-terminus of the Fc domain.

In some embodiments, the hybrid nuclease molecule further includes asecond nuclease domain. In some embodiments, the second nuclease domainis linked to the Fc domain via a second linker domain. In someembodiments, the second linker domain will be at the C-terminus of theFc domain. FIG. 12 shows at least one embodiment of a hybrid nucleasemolecule. In some embodiments, a hybrid nuclease molecule includes asequence shown in Table 2.

In some embodiments, a hybrid nuclease molecule is an RNase molecule orDNase molecule or a multi-enzyme molecule (e.g., both RNase and DNase ortwo RNA or DNA nucleases with different specificity for substrate)attached to an Fc domain that specifically binds to extracellular immunecomplexes. In some embodiments, the Fc domain does not effectively bindFcγ receptors. In one aspect, the hybrid nuclease molecule does noteffectively bind C1q. In other aspects, the hybrid nuclease moleculecomprises an in frame Fc domain from IgG1. In other aspects, the hybridnuclease molecule further comprises mutations in the hinge, CH2, and/orCH3 domains. In other aspects, the mutations are P238S, P331S or N297S,and may include mutations in one or more of the three hinge cysteines.In some such aspects, the mutations in one or more of three hingecysteines can be SCC or SSS. In other aspects, the molecules contain theSCC hinge, but are otherwise wild type for human IgG1 Fc CH2 and CH3domains, and bind efficiently to Fc receptors, facilitating uptake ofthe hybrid nuclease molecule into the endocytic compartment of cells towhich they are bound. In other aspects, the molecule has activityagainst single and/or double-stranded RNA substrates.

In some aspects, the activity of the hybrid nuclease molecule isdetectable in vitro and/or in vivo. In some aspects, the hybrid nucleasemolecule binds to a cell, a malignant cell, or a cancer cell andinterferes with its biologic activity.

In another aspect, a multifunctional RNase molecule is provided that isattached to another enzyme or antibody having binding specificity, suchas an scFv targeted to RNA or a second nuclease domain with the same ordifferent specificities as the first domain.

In another aspect, a multifunctional DNase molecule is provided that isattached to another enzyme or antibody having binding specificity, suchas an scFv targeted to DNA or a second nuclease domain with the same ordifferent specificities as the first domain.

In another aspect, a hybrid nuclease molecule is adapted for preventingor treating a disease or disorder in a mammal by administering an hybridnuclease molecule attached to an Fc region, in a therapeuticallyeffective amount to the mammal in need thereof, wherein the disease isprevented or treated. In other aspects, the disease or disorder is anautoimmune disease or cancer. In some such aspects, the autoimmunedisease is insulin-dependent diabetes mellitus, multiple sclerosis,experimental autoimmune encephalomyelitis, rheumatoid arthritis,experimental autoimmune arthritis, myasthenia gravis, thyroiditis, anexperimental form of uveoretinitis, Hashimoto's thyroiditis, primarymyxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophicgastritis, Addison's disease, premature menopause, male infertility,juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris,pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmunehaemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis,active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerativecolitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis,polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus,or connective tissue disease.

In some embodiments, the targets of the RNase enzyme activity of RNasehybrid nuclease molecules are primarily extracellular, consisting of,e.g., RNA contained in immune complexes with anti-RNP autoantibody andRNA expressed on the surface of cells undergoing apoptosis. In someembodiments, the RNase hybrid nuclease molecule is active in the acidicenvironment of the endocytic vesicles. In some embodiments, an RNasehybrid nuclease molecule includes a wild-type (wt) Fc domain in orderto, e.g, allow the molecule to bind FcR and enter the endocyticcompartment through the entry pathway used by immune complexes. In someembodiments, an RNase hybrid nuclease molecule including a wt Fc domainis adapted to be active both extracellularly and in the endocyticenvironment (where TLR7 can be expressed). In some aspects, this allowsan RNase hybrid nuclease molecule including a wt Fc domain to stop TLR7signaling through previously engulfed immune complexes or by RNAs thatactivate TLR7 after viral infection. In some embodiments, the wt RNaseof an RNase hybrid nuclease molecule is not resistant to inhibition byan RNase cytoplasmic inhibitor. In some embodiments, the wt RNase of anRNase hybrid nuclease molecule is not active in the cytoplasm of a cell.

In some embodiments, a hybrid nuclease molecule including a wt Fc domainis used for therapy of an autoimmune disease, e.g., SLE.

In some embodiments, Fc domain binding to an Fc receptor (FcR) isincreased, e.g., via alterations of glycosylation and/or changes inamino acid sequence. In some embodiments, a hybrid nuclease molecule hasone or more Fc alterations that increase FcR binding.

Alternative ways to construct a hybrid nuclease molecule attached to anFc domain are envisioned. In some embodiments, the domain orientationcan be altered to construct an Ig-RNase molecule or an Ig-DNase moleculeor an RNase-Ig molecule or an RNase-Ig molecule that retains FcR bindingand has active nuclease domains.

In some embodiments, DNase hybrid nuclease molecules include a wt Fcdomain that can allow, e.g., the molecules to undergo endocytosis afterbinding FcR. In some embodiments, the DNase hybrid nuclease moleculescan be active towards extracellular immune complexes containing DNA,e.g., either in soluble form or deposited as insoluble complexes.

In some embodiments, hybrid nuclease molecules include both DNase andRNase. In some embodiments, these hybrid nuclease molecules can improvethe therapy of SLE because they can, e.g., digest immune complexescontaining RNA, DNA, or a combination of both RNA and DNA; and when theyfurther include a wt Fc domain, they are active both extracellularly andin the endocytic compartment where TLR7 and TLR9 can be located.

In some embodiments, linker domains include (gly4ser) 3, 4 or 5 variantsthat alter the length of the linker by 5 amino acid progressions. Inanother embodiment, a linker domain is approximately 18 amino acids inlength and includes an N-linked glycosylation site, which can besensitive to protease cleavage in vivo. In some embodiments, an N-linkedglycosylation site can protect the hybrid nuclease molecules fromcleavage in the linker domain. In some embodiments, an N-linkedglycosylation site can assist in separating the folding of independentfunctional domains separated by the linker domain.

In some embodiments, hybrid nuclease molecules can include both mutantand/or wild type human IgG1 Fc domains. In some embodiments, the hybridnuclease molecules can be expressed from both COS transient and CHOstable transfections. In some embodiments, both the CD80/86 binding andthe RNase activity are preserved in a hybrid nuclease molecule. In someembodiments, hybrid nuclease molecules include DNase1L3-Ig-linker-RNaseconstructs. In some embodiments, a hybrid nuclease molecule includes aDNase1-Ig-linker-RNase construct or an RNase-Ig-linker-DNase construct.In some embodiments, fusion junctions between enzyme domains and theother domains of the hybrid nuclease molecule is optimized.

In some embodiments, hybrid nuclease molecules include DNase-Ig hybridnuclease molecules and/or hybrid DNase-RNase hybrid nuclease molecules.

In some embodiments, a hybrid nuclease molecule includes TREX1. In someembodiments, a TREX1 hybrid nuclease molecule can digest chromatin. Insome embodiments, a TREX1 hybrid nuclease molecule is expressed by acell. In some embodiments, the expressed hybrid nuclease moleculeincludes murine TREX-1 and a murine (wt or mutant) Fc domain. In someembodiments, a 20-25 amino acid (aa) linker domain between TREX1 and theIgG hinge can be required to allow DNase activity. In some embodiments,a hybrid nuclease molecule with a 15 aa linker domain is not active. Insome embodiments, use of the 20 and 25 amino acid linker domains (plus 2or more amino acids to incorporate restriction sites) results infunctional activity as measured by chromatin digestion. In someembodiments, a hydrophobic region of approximately 72 aa can be removedfrom the COOH end of TREX-1 prior to fusion to the Fc domain via thelinker domain. In some embodiments, a 20 amino acid linker domainversion of the hybrid nuclease molecule exhibits high expression levelscompared to controls and/or other hybrid nuclease molecules. In someembodiments, kinetic enzyme assays are used to compare the enzymeactivity of hybrid nuclease molecules and controls in a quantitativemanner.

In some embodiments, further optimization of the fusion junction chosenfor truncation of a TREX1 enzyme can be used to improve expression ofthe hybrid nuclease molecules.

In some embodiments, the hybrid nuclease molecule includes a humanTREX1-linker-Ig Fc domain hybrid nuclease molecule with 20 and/or 25 aalinker domains. In some embodiments, the linker domain(s) are variantsof a (gly4ser)4 or (gly4ser)5 cassette with one or more restrictionsites attached for incorporation into the hybrid nuclease moleculesconstruct. In some embodiments, because of the head-to-tail dimerizationuseful for TREX1 enzyme activity; a flexible, longer linker domain canbe used to facilitate proper folding.

In some embodiments, the hybrid nuclease molecule is a TREX1-tandemhybrid nuclease molecule. In some embodiments, an alternative method forfacilitating head-to-tail folding of TREX1 is to generate aTREX1-TREX1-Ig hybrid hybrid nuclease molecule that incorporates twoTREX1 domains in tandem, followed by a linker domain and an Ig Fcdomain. In some embodiments, positioning of TREX1 cassettes in ahead-to-tail manner can be corrected for head-to tail folding on eitherarm of the immunoenzyme and introduce a single TREX1 functional domaininto each arm of the molecule. In some embodiments, each immunoenzyme ofa hybrid nuclease molecule has two functional TREX1 enzymes attached toa single IgG Fc domain.

In some embodiments, the hybrid nuclease molecule includesTREX1-linker1-Ig-linker2-RNase.

In some embodiments, the hybrid nuclease molecule includesRNase-Ig-linker-TREX1. In some embodiments, cassettes are derived forboth amino and carboxyl fusion of each enzyme for incorporation intohybrid nuclease molecules where the enzyme configuration is reversed. Insome embodiments, the RNase enzyme exhibits comparable functionalactivity regardless of its position in the hybrid nuclease molecules. Insome embodiments, alternative hybrid nuclease molecules can be designedto test whether a particular configuration demonstrates improvedexpression and/or function of the hybrid nuclease molecule components.

In some embodiments, the hybrid nuclease molecule includes 1L3-Ig. Insome embodiments, the 1L3 DNase is constructed from a murine sequenceand expressed. In some embodiments, the enzyme is active. In someembodiments, a murine 1L3 DNase-Ig-RNase hybrid nuclease is constructedand expressed. In some embodiments, the molecule includes human 1L3-Ig,human 1L3-Ig-RNase, and/or human RNase-Ig-1L3.

In some embodiments, the hybrid nuclease molecule includes DNase1-Ig. Insome embodiments, a naturally occurring variant allele, A114F, whichshows reduced sensitivity to actin is included in a DNase1-Ig hybridnuclease molecule. In some embodiments, this mutation is introduced intoa hybrid nuclease molecule to generate a more stable derivative of humanDNase1. In some embodiments, a DNase1-linker-Ig containing a 20 or 25 aalinker domain is made. In some embodiments, hybrid nuclease moleculesinclude RNase-Ig-linker-DNase1 where the DNase1 domain is located at theCOOH side of the Ig Fc domain. In some embodiments, hybrid nucleasemolecules are made that incorporate DNase1 and include:DNase1-linker-Ig-linker2-RNase, and/or RNase-Ig-linker-DNase1.

Another aspect of the present invention is to use gene therapy methodsfor treating or preventing disorders, diseases, and conditions with oneor more hybrid nuclease molecules. The gene therapy methods relate tothe introduction of hybrid nuclease molecule nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe polypeptide or polypeptides of the present invention. This methodcan include introduction of one or more polynucleotides encoding ahybrid nuclease molecule polypeptide of the present inventionoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.

In gene therapy applications, hybrid nuclease molecule genes areintroduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapies where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

Fc Domains

In some embodiments, a hybrid nuclease molecule includes an Fc domain.Fc domains useful for producing the hybrid nuclease molecules of thepresent invention may be obtained from a number of different sources. Inpreferred embodiments, an Fc domain of the hybrid nuclease molecule isderived from a human immunoglobulin. It is understood, however, that theFc domain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e.g. chimpanzee, macaque) species.Moreover, the hybrid nuclease molecule Fc domain or portion thereof maybe derived from any immunoglobulin class, including IgM, IgG, IgD, IgA,and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, andIgG4. In a preferred embodiment, the human isotype IgG1 is used.

A variety of Fc domain gene sequences (e.g. human constant region genesequences) are available in the form of publicly accessible deposits.Constant region domains comprising an Fc domain sequence can be selectedhaving a particular effector function (or lacking a particular effectorfunction) or with a particular modification to reduce immunogenicity.Many sequences of antibodies and antibody-encoding genes have beenpublished and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3sequences, or portions thereof) can be derived from these sequencesusing art recognized techniques. The genetic material obtained using anyof the foregoing methods may then be altered or synthesized to obtainpolypeptides of the present invention. It will further be appreciatedthat the scope of this invention encompasses alleles, variants andmutations of constant region DNA sequences.

Fc domain sequences can be cloned, e.g., using the polymerase chainreaction and primers which are selected to amplify the domain ofinterest. To clone an Fc domain sequence from an antibody, mRNA can beisolated from hybridoma, spleen, or lymph cells, reverse transcribedinto DNA, and antibody genes amplified by PCR. PCR amplification methodsare described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; 4,965,188; and in, e.g., “PCR Protocols: A Guide to Methodsand Applications” Innis et al. eds., Academic Press, San Diego, Calif.(1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.217:270). PCR may be initiated by consensus constant region primers orby more specific primers based on the published heavy and light chainDNA and amino acid sequences. As discussed above, PCR also may be usedto isolate DNA clones encoding the antibody light and heavy chains. Inthis case the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes. Numerous primersets suitable for amplification of antibody genes are known in the art(e.g., 5′ primers based on the N-terminal sequence of purifiedantibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapidamplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods173:33); antibody leader sequences (Larrick et al. 1989 Biochem.Biophys. Res. Commun. 160:1250). The cloning of antibody sequences isfurther described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan.25, 1995, which is incorporated by reference herein.

The hybrid nuclease molecules of the invention may comprise one or moreFc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains). Inone embodiment, the Fc domains may be of different types. In oneembodiment, at least one Fc domain present in the hybrid nucleasemolecule comprises a hinge domain or portion thereof. In anotherembodiment, the hybrid nuclease molecule of the invention comprises atleast one Fc domain which comprises at least one CH2 domain or portionthereof. In another embodiment, the hybrid nuclease molecule of theinvention comprises at least one Fc domain which comprises at least oneCH3 domain or portion thereof. In another embodiment, the hybridnuclease molecule of the invention comprises at least one Fc domainwhich comprises at least one CH4 domain or portion thereof. In anotherembodiment, the hybrid nuclease molecule of the invention comprises atleast one Fc domain which comprises at least one hinge domain or portionthereof and at least one CH2 domain or portion thereof (e.g, in thehinge-CH2 orientation). In another embodiment, the hybrid nucleasemolecule of the invention comprises at least one Fc domain whichcomprises at least one CH2 domain or portion thereof and at least oneCH3 domain or portion thereof (e.g, in the CH2-CH3 orientation). Inanother embodiment, the hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising at least one hinge domain orportion thereof, at least one CH2 domain or portion thereof, and leastone CH3 domain or portion thereof, for example in the orientationhinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge.

In certain embodiments, the hybrid nuclease molecule comprises at leastone complete Fc region derived from one or more immunoglobulin heavychains (e.g., an Fc domain including hinge, CH2, and CH3 domains,although these need not be derived from the same antibody). In otherembodiments, the hybrid nuclease molecule comprises at least twocomplete Fc domains derived from one or more immunoglobulin heavychains. In preferred embodiments, the complete Fc domain is derived froma human IgG immunoglobulin heavy chain (e.g., human IgG1).

In another embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising a complete CH3 domain. Inanother embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising a complete CH2 domain. Inanother embodiment, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain comprising at least a CH3 domain, andat least one of a hinge region, and a CH2 domain. In one embodiment, ahybrid nuclease molecule of the invention comprises at least one Fcdomain comprising a hinge and a CH3 domain. In another embodiment, ahybrid nuclease molecule of the invention comprises at least one Fcdomain comprising a hinge, a CH2, and a CH3 domain. In preferredembodiments, the Fc domain is derived from a human IgG immunoglobulinheavy chain (e.g., human IgG1).

The constant region domains or portions thereof making up an Fc domainof a hybrid nuclease molecule of the invention may be derived fromdifferent immunoglobulin molecules. For example, a polypeptide of theinvention may comprise a CH2 domain or portion thereof derived from anIgG1 molecule and a CH3 region or portion thereof derived from an IgG3molecule. In another example, a hybrid nuclease molecule can comprise anFc domain comprising a hinge domain derived, in part, from an IgG1molecule and, in part, from an IgG3 molecule. As set forth herein, itwill be understood by one of ordinary skill in the art that an Fc domainmay be altered such that it varies in amino acid sequence from anaturally occurring antibody molecule.

In another embodiment, a hybrid nuclease molecule of the inventioncomprises one or more truncated Fc domains that are nonethelesssufficient to confer Fc receptor (FcR) binding properties to the Fcregion. Thus, an Fc domain of a hybrid nuclease molecule of theinvention may comprise or consist of an FcRn binding portion. FcRnbinding portions may be derived from heavy chains of any isotype,including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn bindingportion from an antibody of the human isotype IgG1 is used. In anotherembodiment, an FcRn binding portion from an antibody of the humanisotype IgG4 is used.

In one embodiment, a hybrid nuclease molecule of the invention lacks oneor more constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In a certain embodiments hybrid nucleasemolecules of the invention will lack an entire CH2 domain (ΔCH2constructs). Those skilled in the art will appreciate that suchconstructs may be preferred due to the regulatory properties of the CH2domain on the catabolic rate of the antibody. In certain embodiments,hybrid nuclease molecules of the invention comprise CH2 domain-deletedFc regions derived from a vector (e.g., from IDEC Pharmaceuticals, SanDiego) encoding an IgG1 human constant region domain (see, e.g., WO02/060955A2 and WO02/096948A2). This exemplary vector is engineered todelete the CH2 domain and provide a synthetic vector expressing adomain-deleted IgG1 constant region. It will be noted that theseexemplary constructs are preferably engineered to fuse a binding CH3domain directly to a hinge region of the respective Fc domain.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, a peptidespacer may be placed between a hinge region and a CH2 domain and/orbetween a CH2 and a CH3 domain. For example, compatible constructs couldbe expressed wherein the CH2 domain has been deleted and the remainingCH3 domain (synthetic or unsynthetic) is joined to the hinge region witha 1-20, 1-10, or 1-5 amino acid peptide spacer. Such a peptide spacermay be added, for instance, to ensure that the regulatory elements ofthe constant region domain remain free and accessible or that the hingeregion remains flexible. Preferably, any linker peptide compatible withthe instant invention will be relatively non-immunogenic and not preventproper folding of the Fc.

Changes to Fc Amino Acids

In certain embodiments, an Fc domain employed in a hybrid nucleasemolecule of the invention is altered, e.g., by amino acid mutation(e.g., addition, deletion, or substitution). As used herein, the term“Fc domain variant” refers to an Fc domain having at least one aminoacid substitution as compared to the wild-type Fc from which the Fcdomain is derived. For example, wherein the Fc domain is derived from ahuman IgG1 antibody, a variant comprises at least one amino acidmutation (e.g., substitution) as compared to a wild type amino acid atthe corresponding position of the human IgG1 Fc region.

The amino acid substitution(s) of an Fc variant may be located at aposition within the Fc domain referred to as corresponding to theportion number that that residue would be given in an Fc region in anantibody.

In one embodiment, the Fc variant comprises a substitution at an aminoacid position located in a hinge domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

In certain embodiments, the hybrid nuclease molecules of the inventioncomprise an Fc variant comprising more than one amino acid substitution.The hybrid nuclease molecules of the invention may comprise, forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.Preferably, the amino acid substitutions are spatially positioned fromeach other by an interval of at least 1 amino acid position or more, forexample, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions ormore. More preferably, the engineered amino acids are spatiallypositioned apart from each other by an interval of at least 5, 10, 15,20, or 25 amino acid positions or more.

In certain embodiments, the Fc variant confers an improvement in atleast one effector function imparted by an Fc domain comprising saidwild-type Fc domain (e.g., an improvement in the ability of the Fcdomain to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

The hybrid nuclease molecules of the invention may employ art-recognizedFc variants which are known to impart an improvement in effectorfunction and/or FcR binding. Specifically, a hybrid nuclease molecule ofthe invention may include, for example, a change (e.g., a substitution)at one or more of the amino acid positions disclosed in InternationalPCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1,WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2,WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351 A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091, each of whichis incorporated by reference herein. In one embodiment, the specificchange (e.g., the specific substitution of one or more amino acidsdisclosed in the art) may be made at one or more of the disclosed aminoacid positions. In another embodiment, a different change at one or moreof the disclosed amino acid positions (e.g., the different substitutionof one or more amino acid position disclosed in the art) may be made.

In certain embodiments, a hybrid nuclease molecule of the inventioncomprises an amino acid substitution to an Fc domain which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the antibody. Such hybrid nucleasemolecules exhibit either increased or decreased binding to FcRn whencompared to hybrid nuclease molecules lacking these substitutions and,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such molecules haveuseful applications in methods of treating mammals where long half-lifeof the administered polypeptide is desired, e.g., to treat a chronicdisease or disorder. In contrast, Fc variants with decreased FcRnbinding affinity are expected to have shorter half-lives, and suchmolecules are also useful, for example, for administration to a mammalwhere a shortened circulation time may be advantageous, e.g. for in vivodiagnostic imaging or in situations where the starting polypeptide hastoxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased FcRn binding affinity are also lesslikely to cross the placenta and, thus, are also useful in the treatmentof diseases or disorders in pregnant women. In addition, otherapplications in which reduced FcRn binding affinity may be desiredinclude those applications in which localization the brain, kidney,and/or liver is desired. In one exemplary embodiment, the hybridnuclease molecules of the invention exhibit reduced transport across theepithelium of kidney glomeruli from the vasculature. In anotherembodiment, the hybrid nuclease molecules of the invention exhibitreduced transport across the blood brain barrier (BBB) from the brain,into the vascular space. In one embodiment, a hybrid nuclease moleculewith altered FcRn binding comprises at least one Fc domain (e.g., one ortwo Fc domains) having one or more amino acid substitutions within the“FcRn binding loop” of an Fc domain. Exemplary amino acid substitutionswhich altered FcRn binding activity are disclosed in International PCTPublication No. WO05/047327 which is incorporated by reference herein.

In other embodiments, a hybrid nuclease molecule of the inventioncomprises an Fc variant comprising an amino acid substitution whichalters the antigen-dependent effector functions of the polypeptide, inparticular ADCC or complement activation, e.g., as compared to a wildtype Fc region. In exemplary embodiment, said hybrid nuclease moleculesexhibit altered binding to an Fc gamma receptor (e.g., CD16). Suchhybrid nuclease molecules exhibit either increased or decreased bindingto FcR gamma when compared to wild-type polypeptides and, therefore,mediate enhanced or reduced effector function, respectively. Fc variantswith improved affinity for FcγRs are anticipated to enhance effectorfunction, and such molecules have useful applications in methods oftreating mammals where target molecule destruction is desired. Incontrast, Fc variants with decreased FcγR binding affinity are expectedto reduce effector function, and such molecules are also useful, forexample, for treatment of conditions in which target cell destruction isundesirable, e.g., where normal cells may express target molecules, orwhere chronic administration of the polypeptide might result in unwantedimmune system activation. In one embodiment, the polypeptide comprisingan Fc exhibits at least one altered antigen-dependent effector functionselected from the group consisting of opsonization, phagocytosis,complement dependent cytotoxicity, antigen-dependent cellularcytotoxicity (ADCC), or effector cell modulation as compared to apolypeptide comprising a wild type Fc region.

In one embodiment the hybrid nuclease molecule exhibits altered bindingto an activating FcγR (e.g. FcγI, FcγIIa, or FcγRIIIa). In anotherembodiment, the hybrid nuclease molecule exhibits altered bindingaffinity to an inhibitory FcγR (e.g. FcγRIIb). Exemplary amino acidsubstitutions which altered FcR or complement binding activity aredisclosed in International PCT Publication No. WO05/063815 which isincorporated by reference herein.

A hybrid nuclease molecule of the invention may also comprise an aminoacid substitution which alters the glycosylation of the hybrid nucleasemolecule. For example, the Fc domain of the hybrid nuclease molecule maycomprise an Fc domain having a mutation leading to reduced glycosylation(e.g., N- or O-linked glycosylation) or may comprise an alteredglycoform of the wild-type Fc domain (e.g., a low fucose or fucose-freeglycan). In another embodiment, the hybrid nuclease molecule has anamino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. Exemplary amino acid substitutions which reduce oralter glycosylation are disclosed in International PCT Publication No.WO05/018572 and US Patent Publication No. 2007/0111281, which areincorporated by reference herein.

In other embodiments, a hybrid nuclease molecule of the inventioncomprises at least one Fc domain having engineered cysteine residue oranalog thereof which is located at the solvent-exposed surface.Preferably the engineered cysteine residue or analog thereof does notinterfere with an effector function conferred by the Fc. Morepreferably, the alteration does not interfere with the ability of the Fcto bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) or complementproteins (e.g. C1q), or to trigger immune effector function (e.g.,antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC)). In preferred embodiments, thehybrid nuclease molecules of the invention comprise an Fc domaincomprising at least one engineered free cysteine residue or analogthereof that is substantially free of disulfide bonding with a secondcysteine residue. Any of the above engineered cysteine residues oranalogs thereof may subsequently be conjugated to a functional domainusing art-recognized techniques (e.g., conjugated with a thiol-reactiveheterobifunctional linker).

In one embodiment, the hybrid nuclease molecule of the invention maycomprise a genetically fused Fc domain having two or more of itsconstituent Fc domains independently selected from the Fc domainsdescribed herein. In one embodiment, the Fc domains are the same. Inanother embodiment, at least two of the Fc domains are different. Forexample, the Fc domains of the hybrid nuclease molecules of theinvention comprise the same number of amino acid residues or they maydiffer in length by one or more amino acid residues (e.g., by about 5amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about10 residues, about 15 residues, about 20 residues, about 30 residues,about 40 residues, or about 50 residues). In yet other embodiments, theFc domains of the hybrid nuclease molecules of the invention may differin sequence at one or more amino acid positions. For example, at leasttwo of the Fc domains may differ at about 5 amino acid positions (e.g.,1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15positions, about 20 positions, about 30 positions, about 40 positions,or about 50 positions).

Linker Domains

In some embodiments, a hybrid nuclease molecule includes a linkerdomain. In some embodiments, a hybrid nuclease molecule includes aplurality of linker domains. In some embodiments, the linker domain is apolypeptide linker. In certain aspects, it is desirable to employ apolypeptide linker to fuse one or more Fc domains to one or morenuclease domains to form a hybrid nuclease molecule.

In one embodiment, the polypeptide linker is synthetic. As used hereinthe term “synthetic” with respect to a polypeptide linker includespeptides (or polypeptides) which comprise an amino acid sequence (whichmay or may not be naturally occurring) that is linked in a linearsequence of amino acids to a sequence (which may or may not be naturallyoccurring) (e.g., an Fc domain sequence) to which it is not naturallylinked in nature. For example, the polypeptide linker may comprisenon-naturally occurring polypeptides which are modified forms ofnaturally occurring polypeptides (e.g., comprising a mutation such as anaddition, substitution or deletion) or which comprise a first amino acidsequence (which may or may not be naturally occurring). The polypeptidelinkers of the invention may be employed, for instance, to ensure thatFc domains are juxtaposed to ensure proper folding and formation of afunctional Fc domain. Preferably, a polypeptide linker compatible withthe instant invention will be relatively non-immunogenic and not inhibitany non-covalent association among monomer subunits of a bindingprotein.

In certain embodiments, the hybrid nuclease molecules of the inventionemploy a polypeptide linker to join any two or more domains in frame ina single polypeptide chain. In one embodiment, the two or more domainsmay be independently selected from any of the Fc domains or nucleasedomains discussed herein. For example, in certain embodiments, apolypeptide linker can be used to fuse identical Fc domains, therebyforming a homomeric Fc region. In other embodiments, a polypeptidelinker can be used to fuse different Fc domains (e.g. a wild-type Fcdomain and a Fc domain variant), thereby forming a heteromeric Fcregion. In other embodiments, a polypeptide linker of the invention canbe used to genetically fuse the C-terminus of a first Fc domain (e.g. ahinge domain or portion thereof, a CH2 domain or portion thereof, acomplete CH3 domain or portion thereof, a FcRn binding portion, an FcγRbinding portion, a complement binding portion, or portion thereof) tothe N-terminus of a second Fc domain (e.g., a complete Fc domain).

In one embodiment, a polypeptide linker comprises a portion of an Fcdomain. For example, in one embodiment, a polypeptide linker cancomprise an immunoglobulin hinge domain of an IgG1, IgG2, IgG3, and/orIgG4 antibody. In another embodiment, a polypeptide linker can comprisea CH2 domain of an IgG1, IgG2, IgG3, and/or IgG4 antibody. In otherembodiments, a polypeptide linker can comprise a CH3 domain of an IgG1,IgG2, IgG3, and/or IgG4 antibody. Other portions of an immunoglobulin(e.g. a human immunoglobulin) can be used as well. For example, apolypeptide linker can comprise a CH1 domain or portion thereof, a CLdomain or portion thereof, a VH domain or portion thereof, or a VLdomain or portion thereof. Said portions can be derived from anyimmunoglobulin, including, for example, an IgG1, IgG2, IgG3, and/or IgG4antibody.

In exemplary embodiments, a polypeptide linker can comprise at least aportion of an immunoglobulin hinge region. In one embodiment, apolypeptide linker comprises an upper hinge domain (e.g., an IgG1, anIgG2, an IgG3, or IgG4 upper hinge domain). In another embodiment, apolypeptide linker comprises a middle hinge domain (e.g., an IgG1, anIgG2, an IgG3, or an IgG4 middle hinge domain). In another embodiment, apolypeptide linker comprises a lower hinge domain (e.g., an IgG1, anIgG2, an IgG3, or an IgG4 lower hinge domain).

In other embodiments, polypeptide linkers can be constructed whichcombine hinge elements derived from the same or different antibodyisotypes. In one embodiment, the polypeptide linker comprises a chimerichinge comprising at least a portion of an IgG1 hinge region and at leasta portion of an IgG2 hinge region. In one embodiment, the polypeptidelinker comprises a chimeric hinge comprising at least a portion of anIgG1 hinge region and at least a portion of an IgG3 hinge region. Inanother embodiment, a polypeptide linker comprises a chimeric hingecomprising at least a portion of an IgG1 hinge region and at least aportion of an IgG4 hinge region. In one embodiment, the polypeptidelinker comprises a chimeric hinge comprising at least a portion of anIgG2 hinge region and at least a portion of an IgG3 hinge region. In oneembodiment, the polypeptide linker comprises a chimeric hinge comprisingat least a portion of an IgG2 hinge region and at least a portion of anIgG4 hinge region. In one embodiment, the polypeptide linker comprises achimeric hinge comprising at least a portion of an IgG1 hinge region, atleast a portion of an IgG2 hinge region, and at least a portion of anIgG4 hinge region. In another embodiment, a polypeptide linker cancomprise an IgG 1 upper and middle hinge and a single IgG3 middle hingerepeat motif. In another embodiment, a polypeptide linker can comprisean IgG4 upper hinge, an IgG1 middle hinge and a IgG2 lower hinge.

In another embodiment, a polypeptide linker comprises or consists of agly-ser linker. As used herein, the term “gly-ser linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser linker comprises an amino acid sequence of the formula(Gly₄Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Apreferred gly/ser linker is (Gly₄Ser)4. Another preferred gly/ser linkeris (Gly₄Ser)3. Another preferred gly/ser linker is (Gly₄Ser)5. Incertain embodiments, the gly-ser linker may be inserted between twoother sequences of the polypeptide linker (e.g., any of the polypeptidelinker sequences described herein). In other embodiments, a gly-serlinker is attached at one or both ends of another sequence of thepolypeptide linker (e.g., any of the polypeptide linker sequencesdescribed herein). In yet other embodiments, two or more gly-ser linkerare incorporated in series in a polypeptide linker. In one embodiment, apolypeptide linker of the invention comprises at least a portion of anupper hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4molecule), at least a portion of a middle hinge region (e.g., derivedfrom an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of gly/seramino acid residues (e.g., a gly/ser linker such as (Gly₄Ser)n).

In one embodiment, a polypeptide linker of the invention comprises anon-naturally occurring immunoglobulin hinge region domain, e.g., ahinge region domain that is not naturally found in the polypeptidecomprising the hinge region domain and/or a hinge region domain that hasbeen altered so that it differs in amino acid sequence from a naturallyoccurring immunoglobulin hinge region domain. In one embodiment,mutations can be made to hinge region domains to make a polypeptidelinker of the invention. In one embodiment, a polypeptide linker of theinvention comprises a hinge domain which does not comprise a naturallyoccurring number of cysteines, i.e., the polypeptide linker compriseseither fewer cysteines or a greater number of cysteines than a naturallyoccurring hinge molecule.

In other embodiments, a polypeptide linker of the invention comprises abiologically relevant peptide sequence or a sequence portion thereof.For example, a biologically relevant peptide sequence may include, butis not limited to, sequences derived from an anti-rejection oranti-inflammatory peptide. Said anti-rejection or anti-inflammatorypeptides may be selected from the group consisting of a cytokineinhibitory peptide, a cell adhesion inhibitory peptide, a thrombininhibitory peptide, and a platelet inhibitory peptide. In a onepreferred embodiment, a polypeptide linker comprises a peptide sequenceselected from the group consisting of an IL-1 inhibitory or antagonistpeptide sequence, an erythropoietin (EPO)-mimetic peptide sequence, athrombopoietin (TPO)-mimetic peptide sequence, G-CSF mimetic peptidesequence, a TNF-antagonist peptide sequence, an integrin-binding peptidesequence, a selectin antagonist peptide sequence, an anti-pathogenicpeptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptidesequence, a calmodulin antagonist peptide sequence, a mast cellantagonist, a SH3 antagonist peptide sequence, an urokinase receptor(UKR) antagonist peptide sequence, a somatostatin or cortistatin mimeticpeptide sequence, and a macrophage and/or T-cell inhibiting peptidesequence. Exemplary peptide sequences, any one of which may be employedas a polypeptide linker, are disclosed in U.S. Pat. No. 6,660,843, whichis incorporated by reference herein.

It will be understood that variant forms of these exemplary polypeptidelinkers can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequenceencoding a polypeptide linker such that one or more amino acidsubstitutions, additions or deletions are introduced into thepolypeptide linker. For example, mutations may be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Polypeptide linkers of the invention are at least one amino acid inlength and can be of varying lengths. In one embodiment, a polypeptidelinker of the invention is from about 1 to about 50 amino acids inlength. As used in this context, the term “about” indicates +/− twoamino acid residues. Since linker length must be a positive integer, thelength of from about 1 to about 50 amino acids in length, means a lengthof from 1 to 48-52 amino acids in length. In another embodiment, apolypeptide linker of the invention is from about 10-20 amino acids inlength. In another embodiment, a polypeptide linker of the invention isfrom about 15 to about 50 amino acids in length.

In another embodiment, a polypeptide linker of the invention is fromabout 20 to about 45 amino acids in length. In another embodiment, apolypeptide linker of the invention is from about 15 to about 25 aminoacids in length. In another embodiment, a polypeptide linker of theinvention is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more amino acids in length.

Polypeptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

Nuclease Domains

In certain aspects, a hybrid nuclease molecule includes a nucleasedomain. Accordingly, the hybrid nuclease molecules of the inventiontypically comprise at least one nuclease domain and at least one linkedFc domain. In certain aspects, a hybrid nuclease molecule includes aplurality of nuclease domains.

In some embodiments, a nuclease domain is DNase. In some embodiments,the DNase is a Type I secreted DNase. In some embodiments, the DNase isDNase 1 and/or a DNase 1-like (DNaseL) enzyme, 1-3. In some embodiments,the DNase is TREX1.

In some embodiments, a nuclease domain is an RNase. In some embodiments,the RNase is an extracellular or secretory RNase of the RNase Asuperfamily, e.g., RNase A.

In one embodiment, the nuclease domain is operably linked (e.g.,chemically conjugated or genetically fused (e.g., either directly or viaa polypeptide linker)) to the N-terminus of an Fc domain. In anotherembodiment, the nuclease domain is operably linked (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via apolypeptide linker)) to the C-terminus of an Fc domain. In otherembodiments, a nuclease domain is operably linked (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via apolypeptide linker)) via an amino acid side chain of an Fc domain. Incertain exemplary embodiments, the nuclease domain is fused to an Fcdomain via a human immunoglobulin hinge domain or portion thereof.

In certain embodiments, the hybrid nuclease molecules of the inventioncomprise two or more nuclease domains and at least one Fc domain. Forexample, nuclease domains may be operably linked to both the N-terminusand C-terminus of an Fc domain. In other exemplary embodiments, nucleasedomains may be operably linked to both the N- and C-terminal ends ofmultiple Fc domains (e.g., two, three, four, five, or more Fc domains)which are linked together in series to form a tandem array of Fcdomains.

In other embodiments, two or more nuclease domains are linked to eachother (e.g., via a polypeptide linker) in series, and the tandem arrayof nuclease domains is operably linked (e.g., chemically conjugated orgenetically fused (e.g., either directly or via a polypeptide linker))to either the C-terminus or the N-terminus of a Fc domain or a tandemarray of Fc domains. In other embodiments, the tandem array of nucleasedomains is operably linked to both the C-terminus and the N-terminus ofa Fc domain or a tandem array of Fc domains.

In other embodiments, one or more nuclease domains may be insertedbetween two Fc domains. For example, one or more nuclease domains mayform all or part of a polypeptide linker of a hybrid nuclease moleculeof the invention.

Preferred hybrid nuclease molecules of the invention comprise at leastone nuclease domain (e.g., RNase or DNase), at least one linker domain,and at least one Fc domain.

In certain embodiments, the hybrid nuclease molecules of the inventionhave at least one nuclease domain specific for a target molecule whichmediates a biological effect.

In another embodiment, binding of the hybrid nuclease molecules of theinvention to a target molecule (e.g. DNA or RNA) results in thereduction or elimination of the target molecule, e.g., from a cell, atissue, or from circulation.

In certain embodiments, the hybrid nuclease molecules of the inventionmay comprise two or more nuclease domains. In one embodiment, thenuclease domains are identical, e.g., RNase and RNase, or TREX1 andTREX1. In another embodiment, the nuclease domains are different, e.g.,DNase and RNase.

In other embodiments, the hybrid nuclease molecules of the invention maybe assembled together or with other polypeptides to form bindingproteins having two or more polypeptides (“multimers”), wherein at leastone polypeptide of the multimer is a hybrid nuclease molecule of theinvention. Exemplary multimeric forms include dimeric, trimeric,tetrameric, and hexameric altered binding proteins and the like. In oneembodiment, the polypeptides of the multimer are the same (ie. homomericaltered binding proteins, e.g. homodimers, homotetramers). In anotherembodiment, the polypeptides of the multimer are different (e.g.heteromeric).

Methods of Making Hybrid Nuclease Molecules

The hybrid nuclease molecules of this invention largely may be made intransformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the peptides could be excised from DNA usingsuitable restriction enzymes. Alternatively, the DNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of affecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal nuclease domains, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides. Compounds that contain derivatized peptides orwhich contain non-peptide groups may be synthesized by well-knownorganic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Pharmaceutical Compositions and Therapeutic Methods of Use

In certain embodiments, a hybrid nuclease molecule is administeredalone. In certain embodiments, a hybrid nuclease molecule isadministered prior to the administration of at least one othertherapeutic agent. In certain embodiments, a hybrid nuclease molecule isadministered concurrent with the administration of at least one othertherapeutic agent. In certain embodiments, a hybrid nuclease molecule isadministered subsequent to the administration of at least one othertherapeutic agent. In other embodiments, a hybrid nuclease molecule isadministered prior to the administration of at least one othertherapeutic agent. As will be appreciated by one of skill in the art, insome embodiments, the hybrid nuclease molecule is combined with theother agent/compound. In some embodiments, the hybrid nuclease moleculeand other agent are administered concurrently. In some embodiments, thehybrid nuclease molecule and other agent are not administeredsimultaneously, with the hybrid nuclease molecule being administeredbefore or after the agent is administered. In some embodiments, thesubject receives both the hybrid nuclease molecule and the other agentduring a same period of prevention, occurrence of a disorder, and/orperiod of treatment.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. In certainembodiments, the combination therapy comprises nuclease molecule, incombination with at least one other agent. Agents include, but are notlimited to, in vitro synthetically prepared chemical compositions,antibodies, antigen binding regions, and combinations and conjugatesthereof. In certain embodiments, an agent can act as an agonist,antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a hybrid nuclease molecule together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a hybrid nuclease molecule and a therapeuticallyeffective amount of at least one additional therapeutic agent, togetherwith a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In some embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose.

In certain embodiments, a hybrid nuclease molecule and/or a therapeuticmolecule is linked to a half-life extending vehicle known in the art.Such vehicles include, but are not limited to, polyethylene glycol,glycogen (e.g., glycosylation of the hybrid nuclease molecule), anddextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082, now U.S. Pat. No. 6,660,843 and published PCT ApplicationNo. WO 99/25044, which are hereby incorporated by reference for anypurpose.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which can further include sorbitolor a suitable substitute therefore. In certain embodiments, acomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agents, can be prepared for storage bymixing the selected composition having the desired degree of purity withoptional formulation agents (Remington's Pharmaceutical Sciences, supra)in the form of a lyophilized cake or an aqueous solution. Further, incertain embodiments, a composition comprising a hybrid nucleasemolecule, with or without at least one additional therapeutic agent, canbe formulated as a lyophilizate using appropriate excipients such assucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired hybridnuclease molecule, with or without additional therapeutic agents, in apharmaceutically acceptable vehicle. In certain embodiments, a vehiclefor parenteral injection is sterile distilled water in which a hybridnuclease molecule, with or without at least one additional therapeuticagent, is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that canprovide for the controlled or sustained release of the product which canthen be delivered via a depot injection. In certain embodiments,hyaluronic acid can also be used, and can have the effect of promotingsustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredmolecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, a hybrid nuclease molecule, withor without at least one additional therapeutic agent, can be formulatedas a dry powder for inhalation. In certain embodiments, an inhalationsolution comprising a hybrid nuclease molecule, with or without at leastone additional therapeutic agent, can be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions can benebulized. Pulmonary administration is further described in PCTapplication no. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, a hybrid nuclease molecule,with or without at least one additional therapeutic agents, that isadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption of ahybrid nuclease molecule and/or any additional therapeutic agents. Incertain embodiments, diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of a hybrid nuclease molecule, with or without atleast one additional therapeutic agents, in a mixture with non-toxicexcipients which are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form. Incertain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving a hybrid nuclease molecule,with or without at least one additional therapeutic agent(s), insustained- or controlled-delivery formulations. In certain embodiments,techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci.USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agent, to be employed therapeuticallywill depend, for example, upon the therapeutic context and objectives.One skilled in the art will appreciate that the appropriate dosagelevels for treatment, according to certain embodiments, will thus varydepending, in part, upon the molecule delivered, the indication forwhich a hybrid nuclease molecule, with or without at least oneadditional therapeutic agent, is being used, the route ofadministration, and the size (body weight, body surface or organ size)and/or condition (the age and general health) of the patient. In certainembodiments, the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. In certainembodiments, a typical dosage can range from about 0.1 μg/kg to up toabout 100 mg/kg or more, depending on the factors mentioned above. Incertain embodiments, the dosage can range from 0.1 μg/kg up to about 100mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of a hybrid nuclease molecule and/or anyadditional therapeutic agents in the formulation used. In certainembodiments, a clinician will administer the composition until a dosageis reached that achieves the desired effect. In certain embodiments, thecomposition can therefore be administered as a single dose, or as two ormore doses (which may or may not contain the same amount of the desiredmolecule) over time, or as a continuous infusion via an implantationdevice or catheter. Further refinement of the appropriate dosage isroutinely made by those of ordinary skill in the art and is within theambit of tasks routinely performed by them. In certain embodiments,appropriate dosages can be ascertained through use of appropriatedose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising a hybrid nuclease molecule, with or without atleast one additional therapeutic agent, in an ex vivo manner. In suchinstances, cells, tissues and/or organs that have been removed from thepatient are exposed to a pharmaceutical composition comprising a hybridnuclease molecule, with or without at least one additional therapeuticagent, after which the cells, tissues and/or organs are subsequentlyimplanted back into the patient.

In certain embodiments, a hybrid nuclease molecule and/or any additionaltherapeutic agents can be delivered by implanting certain cells thathave been genetically engineered, using methods such as those describedherein, to express and secrete the polypeptides. In certain embodiments,such cells can be animal or human cells, and can be autologous,heterologous, or xenogeneic. In certain embodiments, the cells can beimmortalized. In certain embodiments, in order to decrease the chance ofan immunological response, the cells can be encapsulated to avoidinfiltration of surrounding tissues. In certain embodiments, theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

The hybrid nuclease molecules of the instant invention are particularlyeffective in the treatment of autoimmune disorders or abnormal immuneresponses. In this regard, it will be appreciated that the hybridnuclease molecules of the present invention may be used to control,suppress, modulate, treat, or eliminate unwanted immune responses toboth external and autoantigens. In yet other embodiments thepolypeptides of the present invention may be used to treat immunedisorders that include, but are not limited to, insulin-dependentdiabetes mellitus, multiple sclerosis, experimental autoimmuneencephalomyelitis, rheumatoid arthritis, experimental autoimmunearthritis, myasthenia gravis, thyroiditis, an experimental form ofuveoretinitis, Hashimoto's thyroiditis, primary myxoedema,thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis,Addison's disease, premature menopause, male infertility, juvenilediabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid,sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolyticanaemia, idiopathic leucopenia, primary biliary cirrhosis, activechronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis,Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis,dermatomyositis, discoid LE, systemic lupus erythematosus, or connectivetissue disease.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Construction of RNase-Ig Fusion Genes

Murine RNase 1 was amplified as a full-length cDNA from an EST library(from Dr. C. Raine, Albert Einstein School of Medicine, Bronx, N.Y.) whosent the clone to our laboratory without an MTA. Sequence specific 5′and 3′ primers used were from the published sequences. The sequence ofthe clone was verified by sequencing analysis. The Genebank accessionnumber is NCBI geneID 19752. Full length human RNase 1 was isolated fromrandom primed and oligo dT primed cDNA derived from human pancreas totalRNA (Ambion/Applied Biosystems, Austin, Tex.).

Once a full-length clone was isolated, primers were designed to create afusion gene with the mouse IgG2a (SEQ ID NO:114) or human IgG1 (SEQ IDNO:110) Fc domains. Two different primers were designed for the 5′sequence fused at the amino terminus of the Fc tail; the firstincorporated the native leader peptide from mouse (or human) RNase,while the second attached an AgeI site to the amino terminus of RNase atthe predicted signal peptide cleavage site in order to fuse the RNase toa human VKIII leader peptide that we already had cloned and used forother expression studies. For the murine RNase, the sequence of thefirst primer is:

mribNL5′

30 mer (RNase 5′ with native leader and HindIII+Kozak)

(SEQ ID NO: 1) gTT AAg CTT gCC ACC ATg ggT CTg gAg AAg TCCCTC ATT CTg-3′

The second primer creates a gene fusion junction between an existingleader sequence and the mature sequence at the 5′ end of the RNase, ator near the predicted leader peptide cleavage site.

27 mer (RNase 5′ mature sequence (no leader, with AgeI site)

(SEQ ID NO: 2) 5′-gAT ACC ACC ggT Agg gAA TCT gCA gCA CAg AAg TTT CAg-3′

The sequence of the 3′ primer for fusion to murine IgG2a at the carboxyend of RNase and the amino terminus of the Fc tail is as follows:

mrib3NH2

28 mer (RNase 3′ end with XhoI site for fusion to mIgG2a).

(SEQ ID NO: 3) 5′-ggC TCg AgC ACA gTA gCA TCA AAg tGG ACTggT ACg TAg g-3′

Two more oligos were designed to create an -Ig-RNase fusion gene, wherethe -Ig tail is amino terminal to the RNase enzyme domain.

mrib5X

36mer RNase 5′ end with linker aa and XbaI site for fusion to carboxyend of Fc domain.

(SEQ ID NO: 4) 5′-AAA TCT AgA CCT CAA CCA ggT Agg gAA TCTgCA gCA CAg AAg TTT CAg-3′

mrib3X

31 mer RNase 3′ end with two stop codons and XbaI site for fusion tocarboxy end of Fc domain.

(SEQ ID NO: 5)5′-TCT AgA CTA TCA CAC AgT AgC ATC AAA gTg gAC Tgg TAC gTA g-3′

Example 2 Isolation of Anti-RNA or Anti-DNA scFvs from MonoclonalAntibody Expressing Hybridomas

An anti-RNA hybridoma designated H564 was used to isolate V regionsspecific for RNA. Prior to harvesting, H564 anti-RNA hybridoma cellswere kept in log phase growth for several days in RPMI 1640 media(Invitrogen/Life Technologies, Gaithersburg, Md.) supplemented withglutamine, pyruvate, DMEM non-essential amino acids, andpenicillin-streptomycin. Cells were pelleted by centrifugation from theculture medium, and 2×10⁷ cells were used to prepare RNA. RNA wasisolated from the hybridoma cells using the QIAGEN RNAeasy kit(Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredderaccording to the manufacturer's instructions accompanying the kit. Fourmicrogram (4 μg) of total RNA was used as template to prepare cDNA byreverse transcription. The RNA, 300 ng random primers, and 500 ng OligodT (12-18), and 1 μl 25 mM dNTPs were combined and denatured at 80° C.for 5 minutes prior to addition of enzyme. Superscript III reversetranscriptase (Invitrogen, Life Technologies) was added to the RNA plusprimer mixture in a total volume of 25 μl in the presence of5.times.second strand buffer and 0.1 M DTT provided with the enzyme. Thereverse transcription reaction was allowed to proceed at 50° C. for onehour.

The cDNA generated in the reverse transcriptase reaction was purified byQIAquick PCR purification kits (QIAGEN, Valencia Calif.) and tailed witha poly-G sequence using terminal transferase (Invitrogen, Carlsbad,Calif.) according to manufacturer's instructions. Tailed cDNA was againpurified by QIAquick PCR purification and eluted in 30 ul elution buffer(EB buffer) provided with the kits. Two microliters of tailed cDNA wasused as template along with an anchor-tail 5′ primer containing a poly-Cdomain, and constant region specific, degenerate 3′ primers to amplifyby PCR the variable regions for the light and heavy chain of the H564antibody. The two variable chains were designed with restriction enzymesites so that a scFv could be assembled by three way ligation of the twoV regions to a linker sequence after amplification and restrictionenzyme digestion.

A (gly4ser)4 peptide linker to be inserted between the two V regions wasincorporated by amplification of this linker sequence by overlapextension PCR using overlapping primers encoding the two halves of themolecule. PCR fragments were isolated by agarose gel electrophoresis,fragments isolated by cutting the appropriate bands from the gel andpurifying the amplified DNA using QIAquick gel extraction kits (QIAGEN,Valencia, Calif.). scFv derivatives from the H564 hybridoma wereassembled as VH-linker-VL fusion genes that could be attached at eitherend of a larger -Ig fusion gene. The V.sub.H domain was amplifiedwithout a leader peptide, but included a 5′ AgeI restriction site forfusion to the V.sub.L and a BglII restriction site at the 3′ end forfusion to the linker domain.

The scFv-Ig was assembled by inserting the scFv HindIII-XhoI fragmentinto pDG containing the human IgG1 hinge, CH2, and CH3 regions, whichwas digested with restriction enzymes, HindIII and XhoI. After ligation,the ligation products were transformed into DH5-alpha bacteria. ThescFv-Ig cDNA was subjected to cycle sequencing on a PE 9700 thermocyclerusing a 25-cycle program by denaturing at 96° C. for 10 seconds,annealing at 50° C. for 30 seconds, and extending at 72° C. for 4minutes. The sequencing primers were pDG forward and reverse primers andan internal primer that annealed to the CH2 domain human in the IgGconstant region portion. Sequencing reactions were performed using theBig Dye Terminator Ready Sequencing Mix v3.1 (PE-Applied Biosystems,Foster City, Calif.) according to the manufacturer's instructions.Samples were subsequently purified using Autoseq G25 columns (GEHealthcare) and the eluates dried in a Savant vacuum dryer, denatured inTemplate Suppression Reagent (PE-ABI), and analyzed on an ABI 310Genetic Analyzer (PE-Applied Biosystems). The sequence was edited,translated, and analyzed using Vector Nti version 10.0(Informax/Invitrogen, North Bethesda, Md.).

Construction of a Human RNaseI-hIgG1 (SEQ ID NO:125-127) fusion gene

Human RNase1 (SEQ ID NO:113) was isolated by PCR amplification fromhuman pancreas total RNA obtained from Ambion/Applied Biosystems(Austin, Tex.). Four microgram (4 μg) of total RNA was used as templateto prepare cDNA by reverse transcription. The RNA, 300 ng randomprimers, and 500 ng Oligo dT (12-18), and 1 ul 25 mM dNTPs were combinedand denatured at 80° C. for 5 minutes prior to addition of enzyme.Superscript III reverse transcriptase (Invitrogen, Life Technologies)was added to the RNA plus primer mixture in a total volume of 25 μl inthe presence of second strand buffer and 0.1 M DTT provided with theenzyme. The reverse transcription reaction was allowed to proceed at 50°C. for one hour. Reactions were further purified by QIAquick PCRpurification columns, and cDNA eluted in 40 microliters EB buffer priorto use in PCR reactions. Two microliters cDNA eluate were added to PCRreactions containing 50 pmol 5′ and 3′ primers specific for humanRNase1, and 45 microliters of PCR high fidelity supermix (Invitrogen,Carlsbad, Calif.) was added to 0.2 ml PCR reaction tubes. PCR reactionswere performed using a C1000 thermal cycler (BioRad, Hercules Calif.).Reactions included an initial denaturation step at 95° C. for 2 minutes,followed by 34 cycles with a 94° C., 30 sec denaturation, 50° C., 30 secannealing, and 68° C., 1 minute extension step, followed by a final 4minute extension at 72° C. Once wild type tails were isolated, thefragments were TOPO cloned into pCR2.1 vectors; DNA prepared using theQIAGEN spin plasmid miniprep kits according to manufacturer'sinstructions. Plasmid DNA was sequenced using ABI Dye Terminator v3.1ready reaction mix according to manufacturer's instructions.

Example 3 Isolation of Human and Mouse -Fc Domains and Introduction ofMutations into the Coding Sequence

For isolation of mouse (SEQ ID NO:114) and human -Fc domains (SEQ IDNO:110), RNA was derived from mouse or human tissue as follows. A singlecell suspension was generated from mouse spleen in RPMI culture media.Alternatively, human PBMCs were isolated from fresh, whole blood usingLymphocyte Separation Media (LSM) Organon Teknika (Durham, N.C.), buffycoats harvested according to manufacturer's directions, and cells washedthree times in PBS prior to use. Cells were pelleted by centrifugationfrom the culture medium, and 2×10⁷ cells were used to prepare RNA. RNAwas isolated from the cells using the QIAGEN RNAeasy kit (Valencia,Calif.) total RNA isolation kit and QIAGEN QIAshredder columns accordingto the manufacturer's instructions accompanying the kits. One microgram(4 μg) of total RNA was used as template to prepare cDNA by reversetranscription. The RNA, 300 ng random primers, and 500 ng Oligo dT(12-18), and 1 μl 25 mM dNTPs were combined and denatured at 80° C. for5 minutes prior to addition of enzyme. Superscript III reversetranscriptase (Invitrogen, Life Technologies) was added to the RNA plusprimer mixture in a total volume of 25 μl in the presence of .secondstrand buffer and 0.1 M DTT provided with the enzyme. The reversetranscription reaction was allowed to proceed at 50° C. for one hour.cDNA was purified using QIAquick (QIAGEN) PCR purification columnsaccording to manufacturer's directions, and eluted in 40 microliters EBbuffer prior to use in PCR reactions.

Wild type mouse and human -Fc domains were isolated by PCR amplificationusing the cDNA described above as template. The following primers wereused for initial amplification of wild type sequences, but incorporatedthe desired mutational changes in the hinge domain:

mahIgG1CH2M: 47 mer (SEQ ID NO: 6)5′-tgtccaccgtgtccagcacctgaactcctgggtggatcgtcagtcttcc-3′hIgG1-5scc: 49 mer (SEQ ID NO: 7)5′-agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgt-3′ mahIgG1S: 51 mer(SEQ ID NO: 8) 5′-tctagattatcatttacccggagacagagagaggctcttctgcgtgtagtg-3′muIgG2aCH2: 58 mer (SEQ ID NO: 9)5′-cctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttcc-3′mIgG2a-5scc: 47 mer (SEQ ID NO: 10)5′-gaagatctcgagcccagaggtcccacaatcaagccctctcctcca-3′ mIgG2a3S: 48 mer(SEQ ID NO: 11) 5′-gtttctagattatcatttacccggagtccgagagaagctcttagtcgt-3′

PCR reactions were performed using a C 1000 thermal cycler (BioRad,Hercules Calif.) or an Eppendorf thermal cycler (ThermoFisherScientific, Houston Tex.). Reactions included an initial denaturationstep at 95° C. for 2 minutes, followed by 34 cycles with a 94° C., 30sec denaturation, 50° C., 30 sec annealing, and 72° C., 1 minuteextension step, followed by a final 4 minute extension at 72° C. Oncewild type tails were isolated, the fragments were TOPO cloned intopCR2.1 vectors, DNA prepared using the QIAGEN spin plasmid miniprep kitsaccording to manufacturer's instructions and clones sequenced using ABIDye Terminator v3.1 sequencing reactions according to manufacturer'sinstructions.

DNA from the correct clones were used as templates in overlap extensionPCRs to introduce mutations at the desired positions in the codingsequence for mouse IgG2a or human -IgG1. PCR reactions were set up usingthe full length wild type clones as template (1 microliter), 50 pmol 5′and 3′ primers to PCR each portion of the -Fc domain up to and includingthe desired mutation site from each direction, and PCR hi fidelitySupermix (Invitrogen, Carlsbad Calif.), in 50 microliter reactionvolumes using a short amplification cycle. As an example of theoverlapping PCR mutagenesis, the primer combination used to introducethe P331S mutation into human -IgG1, was as follows:

A 5′ subfragment was amplified using the full-length wild type clone astemplate, and the 5′ primer was hIgG1-5scc:5′-agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgt-3′ (SEQ ID NO:12),while the 3′ primer was P331AS:5′-gttttctcgatggaggctgggagggctttgttggagacc-3′ (SEQ ID NO:13). A 3′subfragments was amplified using the full-length wild type clone astemplate and the 5′ primer was P331S: 5′aaggtctccaacaaagccctcccagcctccatcgagaaaacaatctcc-3′ (SEQ ID NO:14),while the 3′ primer was mahIgG1S:5′-tctagattatcatttacccggagacagagagaggctettctgcgtgtagtg-3′ (SEQ IDNO:15).

Once subfragments were amplified and isolated by agarose gelelectrophoresis, they were purified by QIAquick gel purification columnsand eluted in 30 microliters EB buffer according to manufacturer'sinstructions. Two rounds of PCR were then performed with the twosubfragments as overlapping templates in new reactions. The cycler waspaused and the 5′ (hIgG1-5scc, see above) and 3′ (mahIgG15, see above)flanking primers were added to the reactions (50 pmol each). PCRamplifications were then carried out for 34 cycles at the conditionsdescribed for the wild type molecules above. Full length fragments wereisolated by gel electrophoresis, and TOPO cloned into pCR2.1 vectors forsequence analysis. Fragments from clones with the correct sequence werethen subcloned into expression vectors for creation of the differenthybrid nuclease molecules described herein.

Example 4 Expression of RNAse-Ig (SEQ ID NO 124, 125, 126, 127, 174(Nucleotide) or 160, 161, 162, 163, 175 (amino acid)), DNAse-Ig (SEQ IDNO: 118, 119, 120, 121, 122, 123, 186 (Nucleotide) or SEQ ID NO 154,155, 156, 157, 158, 159, 187 (Amino Acid)), Multi-Subunit Ig FusionConstructs (SEQ ID NO: 115, 116, 117, 172, 176, 178, 180 (Nucleotide) orSEQ ID NO 151 152, 153, 173, 177, 179, 181 (Amino Acid)), and H564scFv-Ig Fusion Proteins in Stable CHO Cell Lines

This example illustrates expression of the different -Ig fusion genesdescribed herein in eukaryotic cell lines and characterization of theexpressed fusion proteins by SDS-PAGE and by IgG sandwich ELISA.

The -Ig fusion gene fragments with correct sequence were inserted intothe mammalian expression vector pDG, and DNA from positive clones wasamplified using QIAGEN plasmid preparation kits (QIAGEN, Valencia,Calif.). The recombinant plasmid DNA (100 μg) was then linearized in anonessential region by digestion with AscI, purified by phenolextraction, and resuspended in tissue culture media, Excell 302 (Catalog#14312-79P, JRH Biosciences, Lenexa, Kans./SAFC). Cells fortransfection, CHO DG44 cells, were kept in logarithmic growth, and 10⁷cells harvested for each transfection reaction. Linearized DNA was addedto the CHO cells in a total volume of 0.8 ml for electroporation.

Stable production of the -Ig fusion protein was achieved byelectroporation of a selectable, amplifiable plasmid, pDG, containingthe RNase-Ig cDNA under the control of the CMV promoter, into ChineseHamster Ovary (CHO) cells. The pDG vector is a modified version ofpcDNA3 encoding the DHFR selectable marker with an attenuated promoterto increase selection pressure for the plasmid. Plasmid DNA was preparedusing Qiagen maxiprep kits, and purified plasmid was linearized at aunique AscI site prior to phenol extraction and ethanol precipitation.Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrierDNA, and 100 μg each of plasmid and carrier DNA was used to transfect10⁷ CHO DG44 cells by electroporation. Cells were grown to logarithmicphase in Excell 302 media (JRH Biosciences) containing glutamine (4 mM),pyruvate, recombinant insulin, penicillin-streptomycin, and 2×DMEMnonessential amino acids (all from Life Technologies, Gaithersburg,Md.), hereafter referred to as “Excell 302 complete” media. Media foruntransfected cells also contained HT (diluted from a 100× solution ofhypoxanthine and thymidine) (Invitrogen/Life Technologies). Media fortransfections under selection contained varying levels of methotrexate(Sigma-Aldrich) as selective agent, ranging from 50 nM to 1 μM.Electroporations were performed at 280 volts, 950 microFarads.Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 125 cells/well to 2000cells/well. Culture media for cell cloning was Excell 302 complete,containing 50 nM methotrexate. Once clonal outgrowth was sufficient,serial dilutions of culture supernatants from master wells were screenedfor expression of -Ig fusion protein by use of an -IgG sandwich ELISA.Briefly, NUNC immulon II plates were coated overnight at 4° C. with 7.5microgram/ml F(ab′2) goat anti-mouse IgG (KPL Labs, Gaithersburg, Md.)in PBS. Plates were blocked in PBS/3% BSA, and serial dilutions ofculture supernatants incubated at room temperature for 2-3 hours. Plateswere washed three times in PBS/0.05% Tween 20, and incubated withhorseradish peroxidase conjugated F(ab′2) goat anti-mouse IgG2a(Southern Biotechnologies) and goat anti-mouse IgG (KPL) mixed together,each at 1:3500 in PBS/1.0% BSA, for 1-2 hours at room temperature.Plates were washed four times in PBS/0.05% Tween 20, and bindingdetected with SureBlue Reserve, TMB substrate (KPL Labs, Gaithersburg,Md.). Reactions were stopped by addition of equal volume of 1N HCl, andplates read at 450 nM on a Spectramax Pro plate reader (Microdevices,Sunnyvale Calif.). The clones with the highest production of the fusionprotein were expanded into T25 and then T75 flasks to provide adequatenumbers of cells for freezing and for scaling up production of thefusion protein. Production levels were further increased in culturesfrom the four best clones by progressive amplification in methotrexatecontaining culture media. At each successive passage of cells, theExcell 302 complete media contained an increased concentration ofmethotrexate, such that only the cells that amplified the DHFR plasmidcould survive. The production level of the top four unamplified masterwells from the RNaseIg CHO transfectants ranged from 30-50 micrograms/mlculture. The amplified cultures are currently being assayed to determineproduction levels.

Supernatants were collected from CHO cells expressing the RNase-Ig,filtered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.)and were passed over a Protein A-agarose (IPA 300 crosslinked agarose)column (Repligen, Needham, Mass.). The column was washed with columnwash buffer (90 mM Tris-Base, 150 mM NaCl, 0.05% sodium azide, pH 8.7),and bound protein was eluted using 0.1 M citrate buffer, pH 3.0.Fractions were collected and protein concentration was determined at 280nM using a Nanodrop (Wilmington Del.) microsample spectrophotometer, andblank determination using 0.1 M citrate buffer, pH 3.0. Fractionscontaining fusion protein were pooled, and buffer exchange performed byserial spins in PBS using centricon concentrators followed by filtrationthrough 0.2 μm filter devices, to reduce the possibility of endotoxincontamination. An extinction coefficient of 1.05 was determined usingthe protein analysis tools in the Vector Nti Version 10.0 Softwarepackage (Informax, North Bethesda, Md.) and the predicted cleavage sitefrom the online ExPasy protein analysis tools.

Example 5 SDS-PAGE Analysis of RNaseIg Fusion Protein

Purified RNase-Ig (SEQ ID NO:115) was analyzed by electrophoresis onSDS-Polyacrylamide gels. Fusion protein samples were boiled in SDSsample buffer with and without reduction of disulfide bonds and appliedto SDS10% Tris-BIS gels (Catalog #NP0301, Novex, Carlsbad, Calif.). Fivemicrograms of each purified protein was loaded on the gels. The proteinswere visualized after electrophoresis by Coomassie Blue staining (PierceGel Code Blue Stain Reagent, Catalog #24590, Pierce, Rockford, Ill.),and destaining in distilled water. Molecular weight markers wereincluded on the same gel (Kaleidoscope Prestained Standards, Catalog#161-0324, Bio-Rad, Hercules, Calif.). Other samples were run asfollows: Rnase-Ig fusion protein in the sampling buffer (62.5 mMTris-HCl, pH6.8, 2% SDS, 10% glycerol, 0.01% Bromophenol blue) with andwithout 5% 2-mercaptoethanol) was loaded onto the 4-12% pre-cast gel(Bio-RAD). The gel was running at 100 volts until the dye ran off thegel. The gel was stained in the GelCode Blue (Thermo scientific) at roomtemperature overnight and then washed with water.

FIG. 3 shows the RNase-Ig fusion protein compared to mouse IgG. Rnase-Igwas purified from supernatant transfected CHO cells by binding andelution from Protein A sepharose. The SDS-PAGE gel shows that Rnase-Igis approximately 50 kDa when reduced and approximately 110 kDa whennon-reduced.

Example 6 Detection of RNase-Ig in Mouse Sera

SRED Assay

The 2% agarose gel was prepared with distilled water. Poly-IC (Sigma)was dissolved in distilled water at 3 mg/ml and the gel plate wasprepared as follows: 1.5 ml reaction buffer (0.2M Tris-HCl pH 7.0, 40 mMEDTA and 0.1 mg/ml ethidium bromide), 1 ml Poly-IC and 0.5 ml water wereplace in the tube and maintained at 50° C. for 5 min. 3 ml of theagarose (kept at 50° C.) was added to the tube. The mixture wasimmediately poured onto a glass plate. Sampling wells were punched inthe gel. 2 μl of each serum sample was loaded into wells and the gel wasincubated at 37° C. for 4 hours in the moist chamber. Then the gel wasincubated in a buffer (20 mM sodium acetate pH5.2, 20 mg/ml ethidiumbromide) on ice for 30 min. and read under UV.

FIG. 4 shows RNase activity from three mice (410, 413, and 418) afterintravenous injection of Rnase-Ig fusion protein (SEQ ID NO:150)(purified in this experiment from supernatant of transfected COS cellsby binding and elution from protein A sepharose). A standard was used inthe top row. Notice a second injection for mouse 410 (see arrow) after 2weeks. 2 μl serum from each of three mice was loaded on 1% agarose gelcontaining 0.5 mg/ml poly-C. The gel was incubated for 4 hours in amoist chamber at 37° C., and then immersed in a buffer containing 20 mMsodium acetate and 20 ug/ml ethidium bromide for 30 min. The RNaseactivity is reflected by the size and intensity around the central well.This data shows that the RNase-Ig fusion protein has an extendedhalf-life in mouse serum.

Example 7 Use of an Anti-RNA ELISA to Measure RNA Specific Antibodies inMouse Sera

A 96-well plate (Nunc, Thermal fisher scientific) was coated with 50μg/ml of Poly-L-Lysine (Sigma) overnight. After washing five times withPBS containing 0.05% Tween, the plate was coated with 10 μg/ml of yeastRNA in PBS at 4° C. overnight. After washing five times, the plate wasblocked with PBS containing 1% BSA at room temperature for 2 hours.Serum samples at 1:50 dilution were added to the plate and incubated at4° C. overnight. Hybridoma H564 (anti-RNA) culture medium was used asstandard, using two-fold serial dilutions starting at 1:300. Detectionantibody was anti-mouse IgG conjugated with alkaline phosphatase(Jackson Lab), and was added to the plate at 1:5000 for 1 hour at roomtemperature. Phosphatase substrate (Sigma) was dissolved in developingbuffer (ThermoFisher Scientific) and added to the plate at 50 μl/well.Samples were read at 405 nm using a Spectramax Plus plate reader(Microdevices, Sunnyvale, Calif.).

FIG. 5 shows the results from the anti-RNA Antibody ELISA titer beforeand after intravenous injection of RNase-Ig fusion protein (SEQ IDNO:150) from mouse 410. The pre-coated Poly-L-lysine (50 μg/ml) platewas coated with 10 ug/ml yeast RNA. Serum (1:50) was loaded on the plateand incubated overnight at 4° C. Detection antibody was anti-mouseIgG-alkaline phosphatase (Jackson Labs) at 1:5000 for 1 hour at roomtemperature, and then phosphatase substrate was added and read at 405nm. The data show that injection of Rnase-Ig caused a reduction in titerof anti-RNA antibody that persisted for over 3 weeks.

FIG. 6 shows the results from the anti-RNA Antibody ELISA titer beforeand after injection of RNase-Ig fusion protein (SEQ ID NO:150) withinthree weeks from mouse 413. The experiment was done as described formouse 410. Titer of anti-RNA antibody was reduced after injection ofRnase-Ig.

Example 8 IFN-Alpha Production by Human PBMCs is Inhibited by RNaseIgAddition to Cultures in Vitro

RNase-Ig (SEQ ID NO:150) addition abolished the induction ofinterferon-α from human peripheral blood mononuclear cells stimulatedusing immune complexes formed with serum from an SLE patient (J11) plusnuclear extract (NE). Briefly, ELISA plates were coated with 50microliters 1:2500 capture antibody (anti-IFN alpha, PBL 21112-1,Piscataway, N.J.), and incubated overnight at 4° C. Plates were washedwith PBS/0.05% Tween 20, blocked in PBS/1% BSA for 2 hours at roomtemperature, washed with PBS/0.05% Tween-20, and incubated with standarddilutions of IFN-alpha, or with serial dilutions of serum samples, andincubated 2 hours at room temperature. Plates were washed and incubatedwith 1:2000 detection antibody (PBL 31101-2, Piscataway, N.J.) in PBS/1%BSA. Plates were washed in PBS/0.05% Tween-20, and incubated with 50microliters donkey anti-rabbit HRP (Jackson Immunoresearch, Westgrove,Pa.) at 1:12,000 in PBS/1% BSA. Plates were washed five times prior toaddition of TMB substrate. Reactions were stopped by addition of ½volume 2N H2SO4, and samples read at 450 nm on a Spectramax Pro platereader (MicroDevices, Sunnyvale, Calif.). The results are shown in FIG.7, which shows RNase-Ig addition abolished the induction of interferon-αfrom human peripheral blood mononuclear cells stimulated using immunecomplexes formed with serum from an SLE patient (J11) plus nuclearextract.

Example 9 Phenotype of TLR7.1xRNaseA Double Transgenic Mice

We have created mice that overexpress RNaseA (RNase Tg). This nucleaseis expressed at high levels in RNase Tg mice (see FIG. 8). We havedeveloped both a single radial diffusion (SRED) method (left panel) anda much more quantitative ELISA to quantify RNase in the serum (see FIG.9). We crossed RNaseA Tg with TLR7.1 Tg mice to create the double Tg(DTg). TLR7.1 mice have 8-16 copies of TLR7 and develop a veryaggressive, rapidly progressive lupus-like disease and start to die at 3mo of age with a median survival of 6 mo. In a preliminary analysis, webled DTg and littermate controls at 3 mo of age to see whether the DTgmice exhibited signs of improvement. As shown in FIG. 8, DTg mice hadvery high levels of RNase in their serum (equivalent to >13 U/ml RNasebased on our standard with specific activity of 993 U/mg). RNaseAconcentration in Tg and DTg mice was also measured by ELISA assay asshown in FIG. 9. The RNase A Tg and TLR7.1XRNaseA Dtg mice have RNase Aserum concentrations between 1-2 ng/ml.

Detailed Method for Rnase A ELISA (Example 9, FIG. 9)

-   1. Coat plate with anti-RnaseA Abcam Ab(ab6610): 2.5-10 ug/ml O/N in    4C.-   2. Wash plate 3 times with 0.05% Tween/1×PBS-   3. Block with 1% BSA in PBS for at least 1 hour-   4. Wash plate 3 times with 0.05% Tween/1×PBS-   5. Load samples. Sample dilutions at 1:50-   6. Incubate Rm Temp for 2 hours-   7. Wash plate 3 times with 0.05% Tween/1×PBS-   8. Prepare dilution of biotin labeled Anti Rnase Ab at dilution of    1:4500 (2.2 ug/ml). Leave RT for 1 hour (Rockland 200-4688: 10    mg/ml).-   9. Wash plate 3 times-   10. Dilute StrepAV HRP (Biolegend 405210) 1:2500. Cover with foil    and leave at RT for 25-30 min.-   11. Wash 6 times, let the liquid sit in wells for at least 30    seconds in between washes.-   12. Add BD OptEIA substrate A+B1:1. Wait until color changes 5-10    min max. Don't let the top well standard go over 1.0. Add 80 ul.    (CatNos: 51-2606KC; ReagentA, 51-2607KC; ReagentB)-   13. Add 40 ul of 1M sulfuric acid to stop reaction

Product/Reagent Information:

RNaseA Ab: ab6610 (90 mg/ml)

ELISA buffer: 1% BSA in PBS

ELISA wash buffer: 0.05% Tween/1×PBS

Anti RNaseA biotin conjugated Ab: Rockland: 200-4688(10 mg/ml)

Strep AV HRP: Biolegend 405210

BD OptEIA reagent A and B: 51-2606KC and 51-2607KC

Example 10 Survival Curves for TLR7.1 Transgenic Mouse Strains

There was a highly significant difference between the DTg and the TLR7.1littermate controls in survival. As shown in FIG. 10, at 10 months, 61%of TLR7.1 mice had died, whereas 31% of DTg mice had died. This datashows that overexpression of RNaseA exerted a strong therapeutic effect.The reasons why TLR7.1 mice die prematurely is not entirely clear,although severe anemia, thrombocytopenia, and glomerulonephritis couldplay a part. To determine whether red cell and platelet counts werepositively impacted by RNaseA expression in the DTg mice, we performedblood counts but found no differences between the TLR7.1 and DTg mice.In contrast, there was a significant improvement in kidneyhistopathology in the DTg mice. We observed decreased deposition of IgGand C3 in DTg mice. PAS staining, which reflects inflammation in themesangium was also reduced in DTg mice compared to TLR7.1 littermatecontrols. When we have now compared macrophage infiltration of thekidneys using anti-MAC-2 (galectin3) antibody (Lyoda et al. Nephrol DialTransplat 22: 3451, 2007), there were many fewer mac-2 positive cells inthe glomeruli of the DTg mice. The results of counting 20 glomeruli permouse in 5 mice in each group revelaed mean+/−SE of 3.8+/−1.1 and1.4+/−0.2 for single versus DTg respectively, p=0.05. In addition, wequantified glomerular tuft size and observed a significant reduction inglomerular tuft size in the DTg mice (179+/−41 versus 128+/−16.8 um2 insingle versus DTg respectively, p=0.037). In summary, TLR7.1XRNaseA DTgmice survive longer than their single Tg TLR7.1 littermates and haveless inflammation and injury in their kidneys.

Example 11 Analysis of IRGs in Spleens of TLR Tg Mice

Analysis of interferon response genes (IRGs) in the spleens of TLR7.1 Tgand TLR7.1 X RNaseA DTg mice mice showed that expression of the IRF7gene was significantly lower in the DTg mice (p=0.03). Some other IRGsincluding MX1 and VIG1 were lower in DTg mice compared to Tg mice, butthe differences were not significant. See FIG. 11. Quantitative PCR wasperformed as follows: total RNA was isolated from mouse spleens usingthe RNeasy mini kit (Qiagen, Valencia, Calif., USA), DNase treated usingTurbo DNA-free (Applied Biosystems, Foster City, Calif., USA) andfirst-strand cDNA was produced with the RNA-to-cDNA kit (AppliedBiosystems) using random primers. The 260/280 was between 1.7 and 2.0for isolated RNA measured with a NanoDrop (Thermo Scientific, Waltham,Mass., USA). cDNA was diluted to an equivalent of 1 ng/ul total RNA and8 ul were used per reaction. Primers for the reference gene (18s) andgenes of interest (GOI) were synthesized (IDT, Coralville, Iowa, USA)and diluted to the appropriate concentrations for qPCR using moleculargrade water. BLAST results of the primers show specific sequencehomology only to the reference gene or GOI. Reactions in duplicate (20ul) were run on an ABI Fast 7500 system using a 1:1 mix of template andprimer to SensiMix SYBR low-ROX master mix (Bioline, London, UK).Relative quantification was calculated using the 2^(−ddCT) method withage matched wild type B6 mice as baseline to determine fold changes foreach GOI. The dissociation curves for the reactions show a single meltpeak for each gene. The standard curve showed similar amplificationefficiencies for each gene and that template concentrations were withinthe linear dynamic range for each of primer set.

Example 12 Structures for Generating Hybrid Nuclease Molecules

Hybrid nuclease molecules were designed to incorporate desiredstructures and functional activity of single enzyme or multi-enzymestructures as modular cassettes with compatible restriction enzyme sitesfor shuttling and domain exchange. The schematic structure of differentembodiments of hybrid nuclease molecules is illustrated in FIG. 12.Primers are shown in Table 1. The nucleotide and amino acid sequences ofrepresentative hybrid nuclease molecules are shown in Table 2.

General Approach for Generation of Hybrid Nuclease Molecules

Human cDNAs were isolated from human pancreas RNA (Ambion) or human PBMCRNA from normal human peripheral blood lymphocytes (approximately5×10e6) using QIAgen RNAeasy kits (Valencia, Calif.) and QIAshredderkits to homogenize cell lysates (Qiagen, Valencia, Calif.). Human PBMCswere isolated from heparinized human blood diluted 1:1 in D-PBS andlayered over LSM Lymphocyte Separation Medium (MP Biomedicals, Irvine,Calif.) Ficoll gradients.

Mouse spleen RNA was isolated using QIAgen RNAeasy kits (Valencia,Calif.) from approximately 5×10e6 splenocytes. Cells were pelleted bycentrifugation from the culture medium, and 5×10e6 cells were used toprepare RNA. RNA was isolated from the cells using the QIAGEN RNAeasykit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredderaccording to the manufacturer's instructions accompanying the kit. Oneto two microgram (1-2 μg) of total RNA was used as template to preparecDNA by reverse transcription. The RNA, 300 ng random primers, and 500ng Oligo dT (12-18), and 1 μl 25 mM dNTPs were combined and denatured at80° C. for 5 minutes prior to addition of enzyme. Superscript IIIreverse transcriptase (Invitrogen, Life Technologies) was added to theRNA plus primer mixture in a total volume of 25 μl in the presence of 5times second strand buffer and 0.1 M DTT provided with the enzyme. Thereverse transcription reaction was allowed to proceed at 50° C. for onehour.

Between 10-100 ng cDNA was used in PCR amplification reactions usingprimers specific for the nuclease gene of interest (RNaseA, RNase1,DNase1, Trex1, DNase1L3, etc.) For initial cloning reactions, primerswere designed to isolate the full length cDNA or truncation productsencoding the gene of interest. Full length or shortened PCR fragmentswere isolated by agarose gel electrophoresis, and purified using QiagenQIAquick columns to remove nucleotides, primers, and unwanted amplifiedproducts. Purified fragments were cloned into pCR2.1 TOPO cloningvectors (Invitrogen, Carlsbad, Calif.) and transformed into TOP 10competent bacteria. Isolated colonies were picked into Luria Broth mediacontaining 50 ug/ml carbenicillin, and grown overnight to isolateplasmids. TOPO clones were screened for inserts of the correct size bydigestion with EcoRI (NEB, Ipswich, Mass.) restriction enzyme andagarose gel electrophoresis of digested fragments. DNA sequence analysisof positive clones was performed with ABI Ready Reaction Mix v 3.1 andanalyzed using an ABI 3730 XL DNA sequencer. Once correct clones wereobtained, further sequence modifications were designed and PCR reactionsperformed to generate the desired alleles or expression cassettes.Truncation products and alleles were generated by PCR mutagenesis usingoverlapping primers for introduction of mutations at specific positionsin the genes. Linkers were synthesized by overlapping PCR using internaloverlapping primers and successive rounds of PCR to attach additionalsequence to each terminus. Hybrid nuclease molecules were assembled as astring of several interchangeable cassettes. Molecules of the preferredembodiment contain a fixed leader peptide, a nuclease cassette, anoptional cassette encoding a choice of several different polypeptidelinkers, an -Ig Fc domain cassette with either a STOP codon or a linkerat the carboxyl end of the CH3 domain, and for resolvICase typemolecules, a second linker cassette, followed by a second nucleasecassette. FIG. 12 illustrate the cassette type structure of these hybridnuclease molecules and examples of potential sequences inserted at eachposition. Once hybrid nuclease molecules were assembled, they weretransferred to a mammalian expression plasmid pDG appropriate fortransient expression in COS7 or other cells and stable expression in CHODG44 cells using selection for DHFR with methotrexate.

Transient Expression of Hybrid Nuclease Molecules

COS-7 cells were transiently transfected with expression vector pDGcontaining hybrid nuclease molecule gene inserts. The day beforetransfection, cells were seeded at 4×10e5 cells per 60 mm dish in 4 mlDMEM (ThermoFisher/Mediatech cell gro)+10% FBS tissue culture media.DMEM basal media was supplemented with 4.5 g/L glucose, sodium pyruvate,L-glutamine 4 mM, and non-essential amino acids. Fetal bovine serum(Hyclone, Logan, Utah ThermoFisher Scientific) was added to media at 10%final volume. Cells were incubated at 37° C., 5% CO2 overnight and wereapproximately 40-80% confluent on the day of transfection. Plasmid DNAwas prepared using Qiagen (Valencia, Calif.) QIAprep miniprep kitsaccording to manufacturer's instructions, and eluted in 50 ul EB buffer.DNA concentrations were measured using a Nanodrop 1000 (Thermo FisherScientific, Wilmington Del.) spectrophotometer. Plasmid DNA wastransfected using Polyfect (Qiagen, Valencia, Calif.) transfectionreagent according to manufacturer's instructions, using 2.5 ug plasmidDNA per 60 mm dish and 15 ul polyfect reagent in 150 ul serum free DMEMtransfection cocktails. After complex formation, reactions were dilutedinto 1 ml cell growth media containing serum and all supplements, andadded drop-wise to the plates containing 3 ml fresh DMEM completeculture media. Transient transfections were incubated for 48-72 hoursprior to harvesting culture supernatants for further analysis.

Generation of Stable CHO DG44 Transfectants Expressing the HybridNuclease Molecules of Interest

Stable production of the hybrid nuclease molecules was achieved byelectroporation of a selectable, amplifiable plasmid, pDG, containingthe nuclease-Ig cDNA under the control of the CMV promoter, into ChineseHamster Ovary (CHO) cells. The pDG vector is a modified version ofpcDNA3 encoding the DHFR selectable marker with an attenuated promoterto increase selection pressure for the plasmid. Plasmid DNA was preparedusing Qiagen maxiprep kits, and purified plasmid was linearized at aunique AscI site prior to phenol extraction and ethanol precipitation.Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrierDNA, and 100 μg each of plasmid and carrier DNA was used to transfect10⁷ CHO DG44 cells by electroporation. Cells were grown to logarithmicphase in Excell 302 media (JRH Biosciences) containing glutamine (4 mM),pyruvate, recombinant insulin, penicillin-streptomycin, and 2×DMEMnonessential amino acids (all from Life Technologies, Gaithersburg,Md.), hereafter referred to as “Excell 302 complete” media. Media foruntransfected cells also contained HT (diluted from a 100× solution ofhypoxanthine and thymidine) (Invitrogen/Life Technologies). Media fortransfections under selection contained varying levels of methotrexate(Sigma-Aldrich) as selective agent, ranging from 50 nM to 1 μM.Electroporations were performed at 280 volts, 950 microFarads.Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 125 cells/well to 2000cells/well. Culture media for cell cloning was Excell 302 complete,containing 50 nM methotrexate. Once clonal outgrowth was sufficient,serial dilutions of culture supernatants from master wells were screenedfor expression of hybrid nuclease molecules by use of an -IgG sandwichELISA. Briefly, NUNC immulon II plates were coated overnight at 4° C.with 7.5 microgram/ml F(ab′2) goat anti-mouse IgG (KPL Labs,Gaithersburg, Md.) or 2 ug/ml goat anti-human or anti-mouse IgG (JacksonImmunoresearch, West Grove Pa.) in PBS. Plates were blocked in PBS/2-3%BSA, and serial dilutions of culture supernatants incubated at roomtemperature for 2-3 hours. Plates were washed three times in PBS/0.05%Tween 20, and incubated with horseradish peroxidase conjugated F(ab′2)goat anti-mouse IgG2a (Southern Biotechnologies) and goat anti-mouse IgG(KPL) mixed together, each at 1:3500 in PBS/1.0% BSA, or in horseradishperoxidase conjugated F(ab′)2 goat anti-human IgG1 (JacksonImmunoresearch, West Grove, Pa.) at 1:2500 for 1-2 hours at roomtemperature. Plates were washed four times in PBS/0.05% Tween 20, andbinding detected with SureBlue Reserve, TMB substrate (KPL Labs,Gaithersburg, Md.). Reactions were stopped by addition of equal volumeof 1N HCl, and plates read at 450 nM on a Spectramax Pro plate reader(Microdevices, Sunnyvale Calif.). The clones with the highest productionof the hybrid nuclease molecule were expanded into T25 and then T75flasks to provide adequate numbers of cells for freezing and for scalingup production of the fusion protein. Production levels were furtherincreased in cultures from the four best clones by progressiveamplification in methotrexate containing culture media. At eachsuccessive passage of cells, the Excell 302 complete media contained anincreased concentration of methotrexate, such that only the cells thatamplified the DHFR plasmid could survive.

Supernatants were collected from CHO cells expressing the hybridnuclease molecule, filtered through 0.2 μm PES express filters (Nalgene,Rochester, N.Y.) and were passed over a Protein A-agarose (IPA 300crosslinked agarose) column (Repligen, Needham, Mass.). The column waswashed with column wash buffer (90 mM Tris-Base, 150 mM NaCl, 0.05%sodium azide, pH 8.7), and bound protein was eluted using 0.1 M citratebuffer, pH 3.0. Fractions were collected and protein concentration wasdetermined at 280 nM using a Nanodrop (Wilmington Del.) microsamplespectrophotometer, and blank determination using 0.1 M citrate buffer,pH 3.0. Fractions containing hybrid nuclease molecules were pooled, andbuffer exchange performed by serial spins in PBS using centriconconcentrators followed by filtration through 0.2 μm filter devices, toreduce the possibility of endotoxin contamination.

Example 13 Analysis of Enzyme Kinetics for hRNase1-G88D-hIgG1[SCCH-P238S-K322S-P331S]

The human RNase1 sequence was isolated from human pancreas RNA by randomprimed cDNA reverse transcription and PCR amplification as described inExample 12 for nuclease molecules. The following primers at 50 pmol perreaction were used from the primer set listed in the PCR primer table.

hRNase5′age: (SEQ ID NO: 16) accggtaaggaatcccgggccaagaaattcc hRNase3′bx:(SEQ ID NO: 17) ctcgagatctgtagagtcctccacagaagcatcaaagtgg

The mutant form of human RNase G88D was created by using the followingtwo primers in PCR and overlap PCR reactions to introduce a mutation atposition 88 that alters the resistance of the enzyme to the cytoplasmicinhibitor.

hRNaseG88D-S: (SEQ ID NO: 18) agactgccgcctgacaaacgactccaggtaccchRNAseG88D-AS: (SEQ ID NO: 19) gggtacctggagtcgtttgtcaggcggcagtct

Both wild type and mutant versions of human RNase1 were isolated andcloned as described for hybrid nuclease molecules above. The wild typesequence was cloned using the first two primers listed above. Once theRNase fragments were TOPO cloned and sequenced, the AgeI-XhoI cassetteswere transferred to the pDG expression vector already containing thehuman VK3LP insert and the human IgG1-WT cassette. Constructs wereverified by digestion, and plasmid DNA prepared for transienttransfections. Once function was confirmed from small scale transienttransfections, the molecules were stably transfected into CHO DG44 inorder to express sufficient quantities for further in vitro analysis.The wild type human RNase1 fusion protein is shown in Table 2,hVK3LP-hRNase1-WT-hIgG1-WT (SEQ ID NO:163). Similarly, wild type humanRNase1 was also expressed as a fusion gene with a (gly4ser)4 (SEQ IDNO:125 or SEQ ID NO:161) or (gly4ser)5 (SEQ ID NO:126 or SEQ ID NO:162)linker domain inserted between the hRNase cassette and the hIgG1 Fcdomain. The G88D mutant of human RNase1 was also expressed as a fusiongene designated hVK3LP-hRNase-G88D-hIgG1-WT (SEQ ID NO:124 or 160) orhIgG1-SCCH-P238S-K322S-P331S (SEQ ID NO:174 or 175), listed in Table 2.

The Lineweaver Burk plot of enzyme kinetics for the mutanthRNase1-G88D-hIgG1[SCCH-P2385-K3225-P331S] (SEQ ID NO:175) is shown inFIG. 13. To further define the functional characteristics of thebivalent RNase-Ig fusion protein, we performed preliminarydeterminations of the Michaelis constant, Km. Enzyme kinetics ofpurified human RNase1-Ig fusion protein was assayed using the RNaseAlert Substrate (Ambion/IDT, San Diego, Calif.) according tomanufacturer's instructions and fluorescence assayed using a SpectramaxM2 microplate reader (Molecular Devices, Sunnyvale, Calif.).Fluorescence data was collected at 30 second intervals over the courseof a 30 minute incubation, and analyzed using SoftmaxPro Software(Molecular Devices) Reaction rates at different substrate concentrationswere measured and the data is shown in the form of a Lineweaver Burkeplot.

Example 14 Analysis of Binding of hRNase1-hIgG to Human Monocytic Lines

Protein A purified hybrid nuclease molecules hRNase1-hIgG1-WT wereincubated with human monocytic cell lines THP-1 or U937 to assess FcRmediated binding of the wild type or mutant Fc containing molecules.FIG. 14 shows the binding pattern of hRNase1-WT-hIgG1-WT (SEQ ID NO:161)to these two cell lines. Cells were incubated with 5 ug/ml purifiedfusion protein in PBS/2% FBS for 45 minutes on ice, washed three timesin PBS/2% FBS, and incubated with FITC-goat anti-human IgG (Fc specific)(Jackson Immunoresearch, West Grove, Pa.) at 1:200 for 45 minutes onice. Cells were washed two times in PBS/2% FBS and analyzed by flowcytometry using a FACS Canto (BD, Franklin Lakes, N.J.) flow cytometer,and FlowJo software (TreeStar, Ashland, Oreg.).

Example 15 IVIg Blocking of hRNase1-hIgG1 Binding to Human MonocyticLines

THP-1 or U937 cells were pre-incubated with IVIg starting at 10 mg/mland performing 10-fold serial dilutions across the wells of a 96 wellplate. Cells (approximately 1×10e6 per well) were incubated on ice for45 minutes. Pre-bound cells were washed twice and AF750 conjugatedhRNase1-WT-hIgG1-WT (SEQ ID NO:161) at approximately 5 ug/ml was addedto each well. Binding reactions were incubated 45 minutes on ice, washedtwice in PBS/2% FBS, and analyzed by flow cytometry as described above.IVIg was able to partially block the binding of the labeled nucleasefusion protein, but even at 10 mg/ml, there was still residual bindingdetectable above background. FIG. 15 shows the blocking activity ofhuman IVIg for binding to U937 and THP-1 cells by hRNase1-WT-hIgG1-WT(SEQ ID NO:161).

Example 16 Trex1-Ig Activity Assay

Murine Trex1 was cloned from mouse cDNA using the primers listed below:

mTrex1-5′age: (SEQ ID NO: 20) accggtatgggctcacagaccctgccccatggtcacamTrex1-3′bx: (SEQ ID NO: 21) ctcgagatctgttgttccagtggtagccggagtgccgtacatg

PCR reactions were performed using 50 pmol each primer in a total volumeof 50 ul, under an amplification profile of 94C 30 sec; 50C 60 sec; 68C90 sec for 35 cycles of amplification. PCR products were cloned into thepCR2.1 vector and TOPO clones screened as previously described forprototype nuclease fusion gene cloning. Once sequence was verified, thecassettes were subcloned into the pDG expression vector fused to themIgG tail or co-cloned with one of the (g4s)n linkers to constructTrex1-lnk molecules with different length linkers. Plasmid isolates weretransiently transfected into COS cells as described and stable CHOtransfectants generated as described for prototype nuclease fusiongenes.

Fusion genes were constructed encoding Trex1Ig as follows: the genesincorporate the human VK3 leader peptide fused to murine Trex1 truncatedat the COOH terminus by 72 amino acids (to remove the intracellularnuclear targeting sequences) fused to a (gly4ser)4 (SEQ ID NO:130) or(gly4ser)5 linker (SEQ ID NO:131), fused to the murine IgG2a/c allelethat incorporates some changes from the IgGc sequence of the Balb/cIgG2a allele.

The exonuclease activities of Trex1-Ig were measured in 30 ul reactionscontaining 20 mM Tris (pH7.5), 5 mM MgCl₂, 2 mM DTT, using a 36-meroligonucleotide as substrate. Incubation reactions were allowed toproceed for 20-30 min at 37° C. Samples were subjected toelectrophoresis on 23% polyacrylamide DNA gels overnight. Gels wereincubated in TBE buffer containing 0.5 ug/ml ethidium bromide. The DNAwas visualized by UV transilluminator, and photographed using a KodakEDAS 290 digital camera equipped with ethidium bromide filters andanalyzed using Kodak Molecular Imaging Software. The trex1 activityassay results for COS produced mTrex1-(g4s)4-mIgG2a-c (SEQ ID NO:166)and mTrex1-(g4s)5-mIgG2a-c (SEQ ID NO:167) are shown in FIG. 16.

Example 17 Western Blot of mTrex1-Ig Single Hybrid Nuclease MoleculesProduced by COS-7 Transient Transfection

COS-7 cells were transiently transfected with plasmids containing hybridnuclease molecules encoding Trex1-Ig as follows: the genes incorporatethe human VK3 leader peptide fused to murine Trex1 truncated at the COOHterminus by 72 amino acids (to remove the nuclear envelope targetingsequences) fused to a (gly4ser)4 or (gly4ser)5 linker, fused to themurine IgG2a/c allele that incorporates some changes from the IgGcsequence from the Balb/c IgG2a allele. COS supernatants were harvestedafter 72 hours and 0.5-1.0 ml samples (depending on the experiment) wereimmunoprecipitated overnight at 4° C. with 100 ul protein A-agarosebeads. Protein A beads were centrifuged and washed twice in PBS prior toresuspending in reducing SDS-PAGE loading buffer. Samples were heattreated at 100° C. for 5 minutes, protein A beads centrifuged to pellet,and sample buffer loaded onto 10% SDS-PAGE gels. Samples wereelectrophoresed at 150 volts for 1.5-2 hours, and gels blotted tonitrocellulose membranes at 30 mAmp for 1 hour. Western blots wereblocked in TBS/5% non-fat milk overnight. Blots were incubated with1:2500 HRP (horseradish peroxidase) conjugated goat anti-mouse IgG2a/c(Fc specific, KPL) for 1.5 hours at room temperature, washed in PBS/0.5%Tween20 five or more times, and blots developed using ECL reagent. FIG.17 shows a Western blot of immunoprecipitates from COS7 culturesupernatants expressing mTrex1-(g4s)4 (SEQ ID NO:166) or (g4s)5-mIgG2a-c(SEQ ID NO:167) fusion proteins.

Example 18 Exonuclease Activity of DNase1L3 μg CHO Derived FusionProtein

DNase1L3 was cloned from mouse spleen cDNA using the following primerpair to clone the mDNase1L3 including its native leader peptidesequence.

mdnase1L3-NL: (SEQ ID NO: 22)GTT AAG CTT GCC ACC ATG TCC CTG CAC CCA GCT TCC CCA CGC CTGMdnase1L3-3bx: (SEQ ID NO: 23)CTC GAG ATC TGA GGA GCG ATT GCC TTT TTT TCT CTT TTT GAG AG

Alternatively, PCR reactions were set up using the following primer pairto attach to the human VK3 leader peptide instead of the native leader.

mdnase1L3-age: (SEQ ID NO: 24)ACC GGT CTA AGG CTC TGC TCC TTC AAT GTG AGG TCC TTT GGA Mdnase1L3-3bx:(SEQ ID NO: 25) CTC GAG ATC TGA GGA GCG ATT GCC TTT TTT TCTCTT TTT GAG AG

PCR reactions were performed using 50 pmol each primer in a total volumeof 50 ul, under an amplification profile of 94C 30 sec; 50C 60 sec; 68C90 sec for 35 cycles of amplification. PCR products were cloned into thepCR2.1 vector and TOPO clones screened as previously described forprototype nuclease fusion gene cloning. Once sequence was verified, thecassettes were subcloned into the pDG expression vector fused to themIgG tail. Plasmid isolates were transiently transfected into COS cellsas described and stable CHO transfectants generated as described forprototype nuclease fusion genes.

The exonuclease activity in protein extracts from DNase1L3Ig (SEQ IDNO:185) CHO clones was measured in 30 ul reactions containing 20 mM Tris(pH7.5), 5 mM MgCl₂, 2 mM DTT, and a substrate. Incubation was 20-30 minat 37° C. Samples were then run on agarose DNA gel overnight. The gelwas incubated in TBE buffer containing Ethidium bromide. The DNA wasvisualized under UV. The results of chromatin digestion analysis areshown in FIG. 18.

Example 19 Dose Titration of Increasing Volumes of CHO Supernatant forExonuclease Activity

FIG. 19 shows titration analysis of the exonuclease digestion patternsobtained from COS supernatants expressing DNase1L3Ig fusion proteins(SEQ ID NO:183 or 185). Nuclear DNA Degradation assays were performed asfollows: HeLa cells were cultured in DMEM media and nuclei from 10e5cells were isolated using NP-40 lysis. Nuclei were diluted into 200 ulreaction buffer containing 10 mM Hepes (pH 7.0), 50 mM NaCl, 2 mM MgCl₂,2 mM CaCl₂, and 40 mM b-glycerophosphate. Nuclei were incubated for 3hours at 37° C. in the volumes of culture supernatant indicated on thefigure from DNase1L3 transfected COS cells. Nuclear DNA was isolatedusing QiAmp blood DNA minikit. DNA was analyzed by 1.5% agarose gelelectrophoresis. For control reactions, heparin was used at 250 i.u./ml,to inhibit nuclease activity.

Example 20 Construction and Expression of DNase1-Ig Single and DualEnzyme Hybrid Nuclease Molecules

Naturally occurring alleles of human DNase1 or DNase1 like moleculeshave been reported. The A114F mutation has been previously reported tooccur in natural variants of human DNAse1 like enzymes, and to result inactin resistance of the enzymes containing this sequence change. SeePan, C Q, Dodge T H, Baker D L, Prince W E, Sinicropi D V, and Lazarus RA. J Biol Chem 273: 18374-18381, (1998); Zhen A, Parmelee D, Hyaw H,Coleman T A, Su K, Zhang J, Gentz R, Ruben S, Rosen C, and Li Y. Biochemand Biophys Res Comm 231: 499-504 (1997); and Rodriguez A M, Rodin D,Nomura H, Morton C C, Weremowicz S, and Schneider M C. Genomics 42:507-513 (1997), all of which are herein incorporated by reference.

Similarly, the G105R mutation has been reported recently as a singlenucleotide polymorphism in the gene encoding human DNAse 1 that ispolymorphic in some or all populations, and that is relevant toautoimmunity. (See Yasuda T, Ueki M, Takeshita H, Fujihara J,Kimura-Kataoka K, Lida R, Tsubota E, Soejima M, Koda Y, Dato H, PanduroA. Int J Biochem Cell Biol 42(7): 1216-1225 (2010), herein incorporatedby reference). Allelic variants at this position resulted in highactivity harboring DNase 1 isoforms relative to wild type. Anothernaturally occurring, polymorphic mutation (R21S) has also been reportedto confer higher activity. (See Yasuda, supra)

SLE patients have been reported to have significantly decreased levelsof DNase1 activity (See Martinez-Valle F, Balada E, Ordi-Ros J,Bujan-Rivas S, Sellas-Fernandez A, Vilardell-Tarres M. Lupus 18(5):418-423 (2009), herein incorporated by reference).

Naturally occurring enzyme variants may thus be less immunogenic whenadministered to patients, since these isoforms occur in the humanpopulation. We reasoned that the combination of the actin resistantproperties of alleles similar to A114F with the increased enzymaticactivity of alleles like G105R would generate novel allelic variants ofhuman DNase1 that might show improved clinical activity in vitro and invivo. To our knowledge, ours is the first report of this new mutant formof DNase1 generated from a combination of two naturally occurringvariants G105R and A114F.

Human DNase 1 was isolated as described previously from human pancreasRNA (Ambion), by random primed cDNA and PCR using the following primersets:

5′hDNase1-age: (SEQ ID NO: 26)GTT ACC GGT CTG AAG ATC GCA GCC TTC AAC ATC CAG 5′hDNase1-bx:(SEQ ID NO: 27) GTT CTC GAG ATC TTT CAG CAT CAC CTC CAC TGG ATA GTG

Alternatively, the 3′ DNase cassettes were amplified by PCR using thefollowing primer pair.

3′hDNase1-RV: (SEQ ID NO: 28)GTT GAT ATC CTG AAG ATC GCA GCC TTC AAC ATC CAG 3′hDNase1-stop:(SEQ ID NO: 29) GTT TCT AGA TTA TCA CTT CAG CAT CAC CTC CAC TGG ATA GTG

PCR reactions were performed using 50 pmol each primer, 2 ul cDNA, in atotal volume of 50 ul using Platinum PCR Supermix as previouslydescribed. The amplification profile was 94C 30 sec; 55C 30 sec; 68C 90sec for 35 cycles.

Once the wild type gene was amplified by PCR, the fragments weresubjected to gel electrophoresis and 850 bp fragments purified byQIAquick column purification. Fragments were cloned into pCR2.1,transformed by TOPO cloning according to manufacturer's instructions asdescribed for the other constructs. Once sequence was verified, PCRprimers were used to generate subfragments containing naturallyoccurring alleles for DNase1 that have been reported to improve specificactivity and improve resistance to the inhibitory activity of actin.These subfragments contained overlapping sequence, permittingamplification of complete DNase1 subclones containing the desiredallelic variations. COS 7 cells were transiently transfected in 60 mmdishes using Polyfect (Qiagen, Valencia, Calif.) transfection reagent.Plasmid DNA was prepared using the Qiagen QIAprep miniprep kitsaccording to manufacturer's instructions. Plasmids were eluted in 50 ulEB buffer. DNA concentration was measured using the Nanodrop and analiquot equivalent to 2.5 ug plasmid DNA used for each transfectionreaction. Each DNaseIg (SEQ ID NOS.: 118, 119, 120, 121, 122 or 123) orRNase-Ig-DNase (SEQ ID NOS.: 115, 116, 117) expression cassette wasinserted into the mammalian expression vector pDG, a derivative ofpcDNA3.1. Transfected cells were incubated for 72 hours at 37° C., 5%CO2 prior to harvest of culture supernatants for further analysis.Culture supernatants were harvested, residual cells centrifuged from thesolution, and the liquid transferred to new tubes.

COS-7 cells were transiently transfected with plasmids containing humanDNase1 wild type (SEQ ID NO:118) or naturally occurring DNase 1 mutantalleles (G105R and/or A114F) (SEQ ID NO:115, 116, or 117) fused to thewild type human IgG1 Fc domain. This hinge-CH2-CH3 cassette contains asingle C→S mutation in the hinge region to eliminate the first cysteinein this domain since it is unpaired due to absence of its pairingpartner present in the light chain of the antibody. In addition, morecomplex multi-nuclease fusion proteins were also expressed from COS celltransient transfections. Western blot analysis was performed onsupernatants from transient transfectants. The molecules shown in FIG.20 contain human DNase1 fused to the human IgG1 wild type Fc domain (SEQID NO:154, 155, 156, or 159) or include human RNase1 (wild type) fusedto the SCC hinge-CH2-CH3 Fc domain of human IgG1, followed by a novellinker containing an N-linked glycosylation site to protect the linkerdomain from protease cleavage, and the wild type (SEQ ID NO:153) ormutant allele (SEQ ID NO:151 or 152) forms of human DNase1 at thecarboxy terminus of the molecule. COS supernatants were harvested after72 hours and 0.5-1.0 ml samples (depending on the experiment) wereimmunoprecipitated overnight at 4° C. with 100 ul protein A-agarosebeads. Protein A beads were centrifuged and washed twice in PBS prior toresuspending in SDS-PAGE loading buffer, for NuPAGE gels—reducing ornonreducing LDS sample buffer. Samples were heated according tomanufacturer's instructions, protein A beads centrifuged to pellet, andsample buffer loaded onto 5-12% NuPAGE gradient gels. Samples wereelectrophoresed at 150 volts for 1.5-2 hours, and gels blotted tonitrocellulose membranes at 30 mAmp for 1 hour. Western blots wereblocked in TBS/5% non-fat milk overnight. Blots were incubated with1:2500 HRP (horseradish peroxidase) conjugated goat anti-human IgG (Fcspecific, Jackson Immunoresearch) or goat anti-mouse IgG for 1.5 hoursat room temperature, washed in PBS/0.5% Tween20 five or more times, andblots developed using ECL reagent.

Example 22 Screening COS Supernatants for Nuclease Enzyme Activity

FIG. 21 shows the results of RNase activity assays (SRED) analysis onharvested COS supernatants expressing hDNAse1Ig and hRNase1-Ig-hDNase1fusion proteins by SRED.

COS supernatants from transient transfections of the hDNaseIg single ormultispecific nucleases were assayed for nuclease activity as follows. A2% agarose gel was prepared with distilled water. Poly-C (Sigma) wasdissolved in distilled water at 3 mg/ml. the gel plate was prepared asfollows: 1.5 ml reaction buffer (0.2M Tris-HCl pH7.0, 40 mM EDTA and 0.1mg/ml Ethidium bromide), 1 ml Poly-C and 0.5 ml water were place in thetube and maintained at 50° C. for 5 min. 3 ml of the agarose (kept at50° C.) was added to the tube. The mixture was immediately poured ontoglass plate. Sampling wells were punched in the gel. Approximately 2 ulof each sample was loaded and the gel was incubated at 37° C. for 4hours in the moist chamber. Then the gel was incubated in a buffer (20mM sodium acetate pH5.2, 20 mg/ml Ethidium bromide) on ice for 30 min.Gels were photographed on a UV transilluminator using a Kodak digitalcamera DC290 system equipped with ethidium bromide filters and analyzedusing Kodak Molecular Imaging software.

FIG. 22 shows a composite figure displaying results of DNase nucleaseactivity assays performed on COS supernatants from transfected cells.Culture supernatants were harvested 72 hours after transfecting thefollowing clones of DNase1 wild type and mutant-Ig fusion proteins.: (1)090210-8=hDNAse1-WT-hIgG1 WT (SEQ ID NO:154); (2)090210-9=hDNase1-G105R; A114F-hIgG1 WT (SEQ ID NO:159); (3)091210-8=hRNase1-WT-hIgG1-WT-DNase1-G105R; A114F (SEQ ID NO:151), and(4) 091210-14=hRNase-WT-hIgG1-WT-DNase1-A114F (SEQ ID NO:152).

The pH of the supernatants was adjusted to 0.0 with bicarbonate bufferto facilitate binding of expressed -Ig fusion proteins to protein Aagarose beads. Panel A. of FIG. 23 shows gel electrophoresis analysis ofplasmid DNA digestion:Protein A agarose slurry (50 ul per sample) waswashed in PBS, and incubated overnight at 4° C. with 100 ul culturesupernatant to immunoprecipitate -Ig fusion proteins. Immunoprecipitateswere washed 4-5 times in 750 ul PBS, centrifuging at approximately 3500rpm followed by aspiration of PBS. Final protein A precipitates wereresuspended in 50 ul reaction buffer containing 20 mM Tris ph7.5. 2 mMCaCl2 and 2 mM MgC12 containing 1.5 ug plasmid DNA (pDG expressionvector). Reactions were incubated for 30 minutes at 37° C., heated at65° C. for 5 min, and the DNA present in reactions analyzed by agarosegel electrophoresis on 1.5% TBE-agarose gels.

Panel B shows the results of a nuclease activity assay performed on thesame culture supernatants using the DNase Alert Kit (IDT/Ambion).Reaction tubes containing lyophilized DNase Alert Substrate (50 pmoles)were resuspended with 5 ul nuclease free ddH2O supplied with the kit, 5ul 10× DNase alert buffer, and 40 ul protein A slurry immunoprecipitatedas follows: For these immunoprecipitations, 50 ul protein A agarosebeads were incubated overnight with 50 ul culture supernatant. Sampleswere then washed 5 times with 0.75 ml PBS. Final protein A precipitateswere resuspended in 80 ul nuclease free ddH2O, and 40 ul of the slurry(one half the precipitate) was transferred to the reaction tubes.Negative controls with mock transfected IP and ddH20 were also set up. Apositive control was also set up containing DNase1 provided with the kit(2 units). Reactions were incubated 1 hour at 37° C., and exposed toshort wave length UV transillumination to visualize fluorescence.Relative amounts of DNA digestion are indicated by degree offluorescence.

Example 22 Examination of mac-2 Positive Cells in DTg Mice

Early lupus mortality is usually due to nephritis or infection resultingfrom immunosuppression to treat nephritis. Therefore, an extremelyimportant outcome for any new therapy is improvement in nephritis. Whilehuman studies are limited to quantitation of proteinuria and creatinine,in mice one can get an accurate assessment of inflammation and damage tothe kidneys by histology and immunocytochemistry. We report that TLR7.1x RNase double transgenic (DTg) mice showed lower anti-RNA antibodies,less B cell activation, fewer immune deposits and less PAS positivestaining glomeruli. We have further compared macrophage infiltration ofthe kidneys using the anti-Mac-2 (galectin3) antibody (Iyoda et al.Nephrol Dial Transplant 22: 3451, 2007). Frozen sections from kidneysobtained from single or double Tg were examined for numbers of Mac-2+macrophages as well as glomerular size as described (Iyoda et al).Twenty randomly selected glomeruli (from the outer to inner side of thekidney) were counted for positive cells. There are many fewer mac-2positive staining cells in the glomeruli of DTg as compared to single Tgmice (data not shown). The results of counting 20 glomeruli per mouse ina pilot study of n=4-5 in each group, revealed mean+/−SE of 3.8+/−1.1and 1.4+/−0.2 for single versus DTg respectively, p=0.05. In addition,we quantified glomerular tuft size and observed a significant reductionin glomerular tuft size in the DTg mice (179.4+/−41 versus 128+/−16.8um2 in single versus DTg respectively, p=0.037).

Example 23 Km of Purified Murine RNaseA-Ig Fusion Protein

To further define the functional characteristics of the bivalentRNase-Ig fusion protein (SEQ ID NO:150), we performed determinations ofthe Michaelis constant, Km. As shown in FIG. 23, the enzyme has a highaffinity with a provisional Km of 280 nM (as a comparision, RNase A hasa Km of 34 nM using polyC as substrate (delCardayre et al, Prot Eng8:261, 1995)). FIG. 23 shows enzyme kinetics that were assayed using theRnase Alert Substrate (Ambion/IDT) and fluorescence was quantified witha Spectramax M2 microplate Reader. Data was analyzed using Softmax Prosoftware (Molecular Devices). Reaction rates at different substrateconcentrations were measured and the data shown as a Lineweaver-Burkplot. The apparent Km, corrected for volume is 280 nM.

Example 24 Analysis of 564Igi Tg Mice for Anti-RNA Antibodies

564 Igi Tg mice: Dr. Imanishi-Kara inserted the rearranged VDJ genesfrom the H564 hybridoma into the endogenous Igh and Igk loci to createthe 564Igi mouse on a B6 background. Sera from these mice stained thecytoplasm and nucleoli of fixed cells indicating a predominant anti-RNAspecificity. Consistent with this finding and of special relevance tothis patent application, antibody production was inhibited when thesemice were made TRL7 deficient indicating that the stimulus for antibodyproduction is indeed RNA. This mouse strain develops late onsetglomerulonephritis. We analyzed the expression of anti-RNA antibodies inmice transgenic for H564 and also double transgenic mice coexpressing564Ig and RNase transgenes. FIG. 24 compares the levels of anti-RNAantibodies in mouse sera at successive intervals as these transgenicmice aged.

See Gavalchin, J., R. A. Seder, and S. K. Datta. 1987. The NZB X SWRmodel of lupus nephritis. I. Cross-reactive idiotypes of monoclonalanti-DNA antibodies in relation to antigenic specificity, charge, andallotype. Identification of interconnected idiotype families inheritedfrom the normal SWR and the autoimmune NZB parents. J. Immunol.138:128-137; and Berland, R., L. Fernandez, E. Kari, J. H. Han, I.Lomakin, S. Akira, H. H. Words, J. F. Kearney, A. A. Ucci, and T.Imanishi-Kari. 2006. Toll-like receptor 7-dependent loss of B celltolerance in pathogenic autoantibody knockin mice. Immunity 25:429-440.

Example 25 In Vitro Assessment of Hybrid Nuclease Molecule BiologicalActivity

One or more hybrid nuclease molecules are purified, e.g., by affinity orion exchange chromatography as previously described in the examplesabove. In some instances the hybrid nuclease molecule is a polypeptide.In some instances, the hybrid nuclease molecule includes one or moresequences from Table 2. In some instances the molecule is SEQ ID NO:161,162, or 163. In some instances the molecule includes SEQ ID NO:145 andSEQ ID NO:149. In some instances the molecule is SEQ ID NO:151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 166, 167, 169,170, 171, 173, 175, 177, 179, 181, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, or 207. The hybrid nuclease molecule can be any of thosedisclosed herein and any that can be constructed from the sequencesdisclosed herein (see Table 2), e.g., by taking a nuclease domain andlinking it to an Fc domain; or, e.g., taking a nuclease domain andlinking it to an Fc domain with a linker domain. Various linker domains(e.g., those described herein) can be used to link the Fc domains and/ornuclease domains. For example, linker domains 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acidsin length can be used. Molecules are assayed for the specific nucleaseactivity in vitro using qualitative assays to verify that they possessthe desired nuclease function. Specific activities are generally thendetermined by fluorescence based kinetic assays utilizing substratessuch as the RNase or DNase Alert Kit reagents, and a fluorescence platereader set to take readings as a function of time. In addition, proteinsolutions are generally checked for endotoxin contamination using acommercially available kits, such as the Pyrotell Limulus AmebocyteLysate (LAL) kit, 0.06 EU/ml detection limit from Cape Cod, Inc. (E.Palmouth, Mass.). Molecules are then assayed using a variety of in vitroassays for biological activity.

One series of in vitro assays will measure the effect of the moleculeson cytokine production by human PBMC in response to various stimuli, inthe presence or absence of the molecules in the cultures. Normal orpatient human PBMC (approximately 1×10e6 cells) are cultured for 24, 48,or 96 hours depending on the assay. PBMC are cultured in the presence ofstimuli such as TLR ligands, costimulatory antibodies, immune complexes,and normal or autoimmune sera. The effects of the molecules on cytokineproduction is measured using commercially available reagents, such asthe antibody pair kits from Biolegend (San Diego, Calif.) for IL-6,IL-8, IL-10, IL-4, IFN-gamma, TNF-alpha. Culture supernatants from invitro cultures are harvested at 24, 48 hours or later time points todetermine the effects of the molecules on cytokine production. IFN-alphaproduction is measured using, e.g., anti-human IFN-alpha antibodies andstandard curve reagents available from PBL interferon source(Piscataway, N.J.). A similar set of assays is performed using humanlymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells,etc.); purified using, e.g., commercially available magnetic bead basedisolation kits available from Miltenyi Biotech (Auburn, Calif.).

In addition, the effect of the molecules on expression of lymphocyteactivation receptors such as CD5, CD23, CD69, CD80, CD86, and CD25 isassessed at various time points after stimulation. PBMC or isolated cellsubpopulations are subjected to multi-color flow cytometry to determinehow these molecules affect the expression of different receptorsassociated with immune cell activation.

Another set of assays will measure the effects of these molecules on theproliferation of different lymphocyte subpopulations in vitro. Theseassays will utilize, e.g., CFDA-SE staining (Invitrogen, Carlsbad,Calif.) of human PBMCs prior to stimulation. CFSE at 5 mM is diluted1:3000 in PBS/0.5% BSA with 10e7-10e8 PBMCS or purified cell subsets andlabeling reactions incubated for 3-4 minutes at 37° C. prior to washingseveral times in RPMI/10% FBS to remove remaining CFSE. CFSE labeledcells are then incubated in co-culture reactions with various stimuli(TLR ligands, costimulatory antibodies, etc.) and the molecules for 4days prior to analysis of cell proliferation by flow cytometry usingdye-conjugated cell subpopulation specific antibodies.

The effect of these molecules on in vitro maturation of monocytes intoDCs and macrophages is also assessed using both normal and patient PBMCsamples.

The effectiveness of a hybrid nuclease molecule is demonstrated bycomparing the results of an assay from cells treated with a hybridnuclease molecule disclosed herein to the results of the assay fromcells treated with control formulations. After treatment, the levels ofthe various markers (e.g., cytokines, cell-surface receptors,proliferation) described above are generally improved in an effectivemolecule-treated group relative to the marker levels existing prior tothe treatment, or relative to the levels measured in a control group.

Example 26 Administration of a Hybrid Nuclease Molecule to a Mammal inNeed Thereof

Mammals (e.g., mice, rats, rodents, humans, guinea pigs) are used in thestudy. Mammals are administered (e.g., intravenously) one or more hybridnuclease molecules comprising one or more sequences from Table 2 or acontrol. In some instances the molecule is SEQ ID NO:161, 162, or 163.In some instances the molecule includes SEQ ID NO:145 and SEQ ID NO:149.In some instances the molecule is SEQ ID NO:151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 166, 167, 169, 170, 171, 173,175, 177, 179, 181, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, or207. The hybrid nuclease molecule can be any of those disclosed hereinand any that can be constructed from the sequences disclosed herein (seeTable 2), e.g., by taking a nuclease domain and linking it to an Fcdomain; or, e.g., taking a nuclease domain and linking it to an Fcdomain with a linker domain. Various linker domains (e.g., thosedescribed herein) can be used to link the Fc domains and/or nucleasedomains. For example, linker domains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids in lengthcan be used. In some instances the hybrid nuclease molecule isformulated a pharmaceutically acceptable carrier. In some instances themolecule is formulated as described in the pharmaceutical compositionssection above. The hybrid nuclease molecule targets RNase and/or DNase.

Multiple rounds of doses are used where deemed useful. Effects onIFN-alpha levels, IFN-alpha response gene levels, autoantibody titers,kidney function and pathology, and/or circulating immune complex levelsare monitored in the mammals. Similar studies are performed withdifferent treatment protocols and administration routes (e.g.,intramuscular administration, etc.). The effectiveness of a hybridnuclease molecule is demonstrated by comparing the IFN-alpha levels,IFN-alpha response gene levels, autoantibody titers, kidney function andpathology, and/or circulating immune complex levels in mammals treatedwith a hybrid nuclease molecule disclosed herein to mammals treated withcontrol formulations.

In an example, a human subject in need of treatment is selected oridentified. The subject can be in need of, e.g., reducing a cause orsymptom of SLE. The identification of the subject can occur in aclinical setting, or elsewhere, e.g., in the subject's home through thesubject's own use of a self-testing kit.

At time zero, a suitable first dose of a hybrid nuclease molecule isadministered to the subject. The hybrid nuclease molecule is formulatedas described herein. After a period of time following the first dose,e.g., 7 days, 14 days, and 21 days, the subject's condition isevaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels. Other relevant criteria can also bemeasured. The number and strength of doses are adjusted according to thesubject's needs.

After treatment, the subject's IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels are lowered and/or improved relativeto the levels existing prior to the treatment, or relative to the levelsmeasured in a similarly afflicted but untreated/control subject.

In another example, a rodent subject in need of treatment is selected oridentified. The identification of the subject can occur in a laboratorysetting or elsewhere.

At time zero, a suitable first dose of a hybrid nuclease molecule isadministered to the subject. The hybrid nuclease molecule is formulatedas described herein. After a period of time following the first dose,e.g., 7 days, 14 days, and 21 days, the subject's condition isevaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels. Other relevant criteria can also bemeasured. The number and strength of doses are adjusted according to thesubject's needs.

After treatment, the subject's IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels are lowered and/or improved relativeto the levels existing prior to the treatment, or relative to the levelsmeasured in a similarly afflicted but untreated/control subject.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

Tables

TABLE 1 Primer Listing for RNase and DNase -Ig Fusion Gene ConstructsSEQ ID NO: Name Sequence — human primers: 30 mahIgG1CH2Mtgtccaccgtgtccagcacctgaactcctgggtggatcgtcagtcttcc 31 huIgG1-H1agatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgt 32 hIgG1-5sccgaagatctcgagcccaaatcttctgacaaaactcacacatgt 33 hIgG1SSSHgttagatctcgagcccaaatcttctgacaaaactcacacatct 34 mahIgG1Stctagattatcatttacccggagacagagagaggctcttctgcgtgtag tg 35 P331Saaggtctccaacaaagccctcccagcctccatcgagaaaacaatctcc 36 P331ASgttttctcgatggaggctgggagggctttgttggagacc 37 5′hrnaseAAG CTT GCC ACC ATG GCT CTG GAG AAG TCT CTT GTC CGG CTC C 38 3′hrnasebxctcgagatctgtagagtcctccacagaagcatcaaagtgg 39 5′hrnaseageaccggtaaggaatcccgggccaagaaattcc 40 3′hRNaseRVgatatcccttccctgggcaaggaatcccgggccaagaaattccag 41 3′hRNase-gtttctagattattaggtagagtcctccacagaagcatcaaagtg stop 42 hdnase1L3-GGT AAG CTT GCC ACC ATG TCA CGG GAG CTG GCC CCA 5NL CTG CTG CTT 43hdnase1L3- CTC GAG ATC TGA GGA GCG TTT GCT CTT TGT TTT CTT 3bx CCT TAG44 hDNase1L3- accggtatgaggatctgctccttcaacgtcaggtcctttgg 5age 455′hDNase1- GTT ACC GGT CTG AAG ATC GCA GCC TTC AAC ATC CAG age 465′hDNase1- GTT CTC GAG ATC TTT CAG CAT CAC CTC CAC TGG ATA bx GTG 473′hDNase1- GTT GAT ATC CTG AAG ATC GCA GCC TTC AAC ATC CAG RV 483′hDNase1- GTT TCT AGA TTA TCA CTT CAG CAT CAC CTC CAC TGG stop ATA GTG49 hDNase1 GAT GGC TGC GAG CCC TGC AGG AAC GAC ACC TTC AAC s105-114CGA GAG CCA TTC ATT GTC AGG TTC 50 hDNase1-GAA CCT GAC AAT GAA TGG CTC TCG GTT GAA GGT GTC as114-105GTT CCT GCA GGG CTC GCA GCC ATC 51 hDNase1-GGA GAA GAA CCT GAC AAT GAA TGG CTC TCG GTT GAA as114 GGT 52 hDNase1-ACC TTC AAC CGA GAG CCA TTC ATT GTC AGG TTC TTC s114 TCC 53 hTrex1-accggtatgggccctggagctcgcagacagggcag 5′age 54 hTrex1-3′bxctcgagatctttggtcctagcagaggctgtgacc 55 hTrex1-5′accggtctcgagatgggccctggagctcgcagacagg AX 56 hTrex1-ctcgagtttggtcctagcagaggctgtgacc 3′xho#2 Murine Primers: 57 mTrex1-accggtatgggctcacagaccctgccccatggtcaca 5′age 58 mTrex1-3′bxctcgagatctgttgttccagtggtagccggagtgccgtacatg 59 mdnase1L3-GTT AAG CTT GCC ACC ATG TCC CTG CAC CCA GCT TCC 5NL CCA CGC CTG 60mdnase1L3- CTC GAG ATC TGA GGA GCG ATT GCC TTT TTT TCT CTT 3bxTTT GAG AG 61 mrib1-NL gTT AAg CTT gCC ACC ATg ggT CTg gAg AAg TCC CTCATT CTg 62 mrib3NH2 ggC TCg AgC ACA gTA gCA TCA AAg tGG ACT ggT ACgTAg g 63 muIgG2aCH2 cctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttcc 64 mIgG2a-5 agatctcgagcccagaggtcccacaatcaagccctctcctccatgcaaatgcc 65 mIgG2a-5scc gaagatctcgagcccagaggtcccacaatcaagccctctcctcca 66muIgG2aSSSH atcaagccctctcctccatctaaatccccagcacctaac 67 mIgG2aKP5agtggcaaggagttcaaatgctcggtcaagaagaaagacctcccagcgt ccatcgag 68 mIgG2aKP3ggttctctcgatggacgctgggaggtctttgttgttgaccgagcatttg aactcc 69 mIgG2a3SgtttctagattatcatttacccggagtccgagagaagctcttagtcgtOther Primers for different tail mutations andfor multispecific fusion genes: 70 hIgGl-3ns-gctagctccgtcgactttacccggagacagagagagg ns 71 K322Sgactggctgaatggcaaggagtacaagtgctcggtctccaacaaagccc tc 72 K322ASgagggctttgttggagaccgagcacttgtaagacttgccattcagccag tc 73 hIgG1N297Sccgcgggaggagcagtacagcagcacgtaccgtgtggtcagcgtc 74 hIgG1N297S3gacgctgaccacacggtacgtgctgctgtactgctcctcccgcgg 75 mIgG2aNSgatatctctagatttacccggagtccgagagaagctcttagtcgt 76 mIgG2a3ns-gatatctccggagtcgactttacccggagtccgagagaagctcttag sal 77 mIgG2N297S5cacaaacccatagagaggattacagcagtactctccgggtggtc 78 mIgG2N297S3gaccacccggagagtactgctgtaatcctctctatgggtttgag 79 80 g4s4clnk3GAT ATC ACC GGT AGA ACC ACC TCC ACC ACT CCC ACC TCC TCC AGT GCC TCC 81g4s4clnk5 GTC GAC TCC GGA GGA GGT GGC TCA GGT GGT GGA GGCAGT GGA GGA GGT GG 82 Nlnkgly5aaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtg 83 Nlnkgly3cccatgatatcctgcacgctggggctgctc 84 hdnase1ageACC GGT ATG AGG ATC TGC TCC TTC AAC GTC AGG TCC TTT GG 85 hdnase1L3-AGA TCT TTA TCA GGA GCG TTT GCT CTT TGT TTT CTT 3S CCT TAG 86 mdnase1L3-TCT AGA TTA TCA GGA GCG ATT GCC TTT TTT TCT CTT 3S TTT GAG AG 87mdnase1L3- ACC GGT CTA AGG CTC TGC TCC TTC AAT GTG AGG TCC age TTT GGA88 mrib-L5′ gAT ACC ACC ggT Agg gAA TCT gCA gCA CAg AAg TTT CAg 89mrib5X AAA TCT AgA CCT CAA CCA ggT Agg gAA TCT gCA gCA CAg AAg TTT CAg90 mrib3X TCT AgA CTA TCA CAC AgT AgC ATC AAA gTg gAC Tgg TAC gTA 91hRNaseG88D- agactgccgcctgacaaacgactccaggtaccc S 92 hRNAseG88D-gggcacctggagtcgtttgtcaggcggcagtct AS 93 g4s5-5-1GGC TCA GGT GGT GGA GGA TCT GGA GGA GGT GGC TCA GGT GGT GGA GGA TCT G 94g4s5-2s GTT AGA TCT CTC CGG AGG AGG TGG CTC AGG TGG TGG AGA ATC TGG A 95g4s5-asxho CTC GAG ACT CCC ACC TCC TCC AGA TCC TCC ACC ACC TGA GCC ACC T96 g4s4-5′ AAA GAT CTC TCC GGA GGA GGT GGC TCA GGT GGT GGAGGA TCT GGA GGA GG 97 g4s4-3′CTC GAG ACC GGT AGA ACC ACC TCC ACC ACT CCC ACC TCC TCC AGA TCC TC 98g4s5-5 GTT AGA TCT CTC CGG AGG AGG TGG CTC A 99 g4s5-3ACC GGT CTC GAG ACT CCC ACC TCC TCC AGA TC

TABLE 2 SEQ ID DESCRIP- NO: TIONSEQUENCE (NUCLEOTIDE SEQUENCES ARE 5′-3′) 100 g4s4lnkagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgag 101 G4S5-1agatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtaccggtctcgag 102 G4S5-2agatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgag 103 3′hRNasegtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatc G88Dccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacgactccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 104 hDNase1-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 3′-tccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatc G104R; A1gccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctg 14Fgacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataatctaga 105 hDNase1-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 3′-WTtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaatgataatctaga 106 hDNase1-gatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 3′A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataatctaga 107 hDNase1-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 5′-tccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatc G105R; A1gccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctg 14Fgacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 108 hDNase1-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 5′-WTtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 109 hDNase1-accggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatg 5′-A114Ftccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgag 110 hIgG1Wtagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaa atgataatctaga 111hDNase1 + gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc VK3LPtggctcccagataccaccggtctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggccagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtga 112 hDNase1Latgtcacgggagctggccccactgctgcttctcctcctctccatccacagcgcc 3ctggccatgaggatctgctccttcaacgtcaggtcctttggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatcaaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatctgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatacaactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcctttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgactatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcctag 113 humanatgggtctggagaagtctcttgtccggctccttctgcttgtcctgatactgctg pancreaticgtgctgggctgggtccagccttccctgggcaaggaatcccgggccaagaaattc ribonu-cagcggcagcatatggactcagacagttcccccagcagcagctccacctactgt cleaseaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgctactgtgtag 114 huVK3LP +gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc mrib1 + mItggctcccagataccaccggtagggaatctgcagcacagaagtttcagcggcag gG2A-C-cacatggatccagatggttcctccatcaacagccccacctactgcaaccaaatg 2Satgaaacgccgggatatgacaaatgggtcatgcaagcccgtgaacaccttcgtgcatgagcccttggcagatgtccaggccgtctgctcccaggaaaatgtcacctgcaagaacaggaagagcaactgctacaagagcagctctgccctgcacatcactgactgccacctgaagggcaactccaagtatcccaactgtgactacaagaccactcaataccagaagcacatcattgtggcctgtgaagggaacccctacgtaccagtccactttgatgctactgtgctcgagcccagaggtctcacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatggtcacatgtgtggtggcggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctggactccgggtaaatgataatctagaa 115 huVK3LP-aagcttgccgccatggaaaccccagcgcagcttctcttcctcctgctactctgg hRNaseWT-ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat hIgGWt-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg NLG-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac hDNase1-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag 105-114aacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggctcggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccagnctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataa tctaga 116huVK3LP- aagcttgccgccatggaaaccccagcgcagcttctcttcctcctgctactctgghRNaseWT- ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat hIgGWT-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg NLG-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac hDNase1-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag 114Faacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataa tctaga 117huVK3LP- aagcttgccgccatggaaaccccagcgcagcttctcttcctcctgctactctgghRNaseWT- ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat hIgGWT-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg NLG-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac hDNase1-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag WTaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgcccaatgccacccccgtcagctacattgcgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaatgataa tctaga 118hVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt (WT)-ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg hIgG1WTagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctccctccgcccccgggtaaatgataatcc aga 119 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt A114F-ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg hIgG1WTagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttagaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatct aga 120 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)4-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac hIgG1WTgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 121 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg (G4S)5-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc 1-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac hIgG1WTgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 122 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)5-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac 2-hIgG1-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg WTtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagcacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 123 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc hIgG1WTgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatct aga 124 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNase(Mtggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag T)-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg hIgG1WTatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacgactccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 125 hVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNase(Wtggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag T)-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg (G4S) 41natgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtg k-cacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgc hIgG1WTaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactcCacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatct aga 126 hVK3LP-gttaagcttaccaccatggaaaccccagcgcagcttctcttcctcctactactc hRNase(Wtggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag T)-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg (G4S)5-atgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtg 2-lnk-cacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgc hIgG1WTaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatccggaggaggtgggagtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaa tgataatctaga 127hVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNase(Wtggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag T)-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg hIgG1WTatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 128 murineatgggctcacagaccctgccccatggtcacatgcagaccctcatcttcttagac Trex1ctggaagccactggcctgccttcgtctcggcccgaagtcacagagctgtgcctg (FL)ctggctgtccacagacgtgctctggagaacacttccatttctcagggacatcca trans-cctccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgct criptccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagct variantgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctc 1cgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgtgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggtgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaaccaacctaaggccacatgctgccacagctactacacccctggccacagccaatggaagtcccagcaatggcaggagcaggcgacctaagagtcctcctccagagaaggtcccagaagccccatcacaggaggggctgctggccccactgagcctgctgaccctcctgaccttggcaatagccactctgcatggactcttcctggcctcacctgggcagtaa 129 mTREX1miatgggctcacagaccctgccccatggtcacatgcagaccctcatcttcttagac necctggaagccactggcctgccttcgtctcggcccgaagtcacagagctgtgcctgctggctgtccacagacgtgctctggagaacacttccatttctcagggacatccacctccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgctccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagctgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctccgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgtgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggtgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaacagatctcgag 130 murineaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg Trex1-ctcccagataccaccggtatgggctcacagaccctgccccatggtcacatgcag (G4S)4-accctcatcttcttagacctggaagccactggcctgccttcgtctcggcccgaa mIgG2a-cgtcacagagctgtgcctgctggctgtccacagacgtgctctggagaacacttccatttctcagggacatccacctccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgctccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagctgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctccgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgcgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggtgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcagggagtggtggaggtggttctaccggtctcgagcccagaggtcccacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatggtcacatgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctcggactccgggtaaatgataatctaga 131 murineaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg Trex1-ctcccagataccaccggtatgggctcacagaccctgccccatggtcacatgcag (G4S)5-accctcatcttcttagacctggaagccactggcctgccttcgtctcggcccgaa mIgG2a-cgtcacagagctgtgcctgctggctgtccacagacgtgctctggagaacacttccatttctcagggacatccacctccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgctccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagctgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctccgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgtgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggtgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgagcccagaggtcccacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcacgatctccctgagccccatggtcacatgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctcggactccgggtaaatgataatctaga 132 NLGgtcgacggcgcggccgccagccccgtgaacgtgagcagccccagcgtgcaggat linker atc 133murine aagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg Trex1-ctcccagataccaccggtatgggctcacagaccctgccccatggtcacatgcag Trex1-accctcatcttcttagacctggaagccactggcctgccttcgtctcggcccgaa (g4S)5-gtcacagagctgtgcctgctggctgtccacagacgtgctctggagaacacttcc mIgG2a-catttctcagggacatccacctccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgctccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagctgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctccgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgtgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggtgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaacagatctcatgggctcacagaccctgccccatggtcacatgcagaccctcatcttcttagacctggaagccactggcctgccttcgtctcggcccgaagtcacagagctgtgcctgctggctgtccacagacgtgctctggagaacacctccatttctcagggacatccacccccagtgcccagaccgccccgtgtggtggacaagctctctctgtgcattgctccagggaaagcctgtagccctggggccagtgagatcacaggtctgagcaaagctgagctggaagtacaggggcgtcaacgcttcgatgacaacctggccatcctgctccgagccttcctgcagcgccagccacagccttgctgccttgtggcacacaacggtgaccgctatgactttcctctgctccagacagagcttgctaggctgagcactcccagtcccctagatggtaccttctgtgtggacagcatcgctgccctaaaggccttggaacaagctagcagcccctcagggaatggttcgaggaaaagctacagcctgggcagcatctacacccgcctgtactggcaagcaccgacagactcacatactgctgaaggcgatgttctaaccctgctcagcatctgtcagtggaagccacaggccctactgcagtgggtggacgaacatgcccggccctttagcaccgtcaagcccatgtacggcactccggctaccactggaacaacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggcccaggtggtggaggatctggaggaggtgggagtctcgagcccagaggtcccacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatggtcacatgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctcggactccgggtaaatgataa tctaga 134huVk3LP- aagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg huTREX1-ctcccagataccaccggtatgggccctggagcccgcagacagggcaggattgtg 72aa-cagggaaggcctgagatgtgcttctgcccaccccctaccccactccctcccctt (g4s)4-cggatcttaacactgggcactcacacacccaccccatgctcctctccaggctca hIgG1-WTgcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 135 huVK3LP-aagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg huTREX1-ctcccagataccaccggtatgggccctggagctcgcagacagggcaggattgtg 72aa-cagggaaggcctgagatgtgcttctgcccaccccctaccccactccctcccctt (g4s)5-cggatcttaacactgggcactcacacacccaccccatgctcctctccaggctca hIgG1-WTgcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 136 g4s4lnk dlsggggsggggsggggsggggstgle137 G4S5-1 dlsggggsggggsggggsggggsggggstgle 138 G4S5-2dlsggggsggggsggggsggggsggggsle 139 hDNase1-dilkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkll 3′-dnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrn G105R; A1dtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgl 14Fedvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 140 hDNase1-dilkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkll 3′-WTdnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 141 hDNase1-dilkiaafniqtfgetkmsnativsyivqilsrydialvqevrdshltavgkll 3′A114Fdnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlk* 142 hDNase1-tglkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkll 5′-dnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcrn G105R; A1dtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgl 14Fedvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdle 143 hDNase1-tglkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkll 5′-WTdnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdle 144 hDNase1-tglkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgkll 5′-A114Fdnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdle 145 hIgG1WTdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 146 hRNase-vdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrr G88D-3′rnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltndsrypncayrtspkerhiivacegspyvpvhfdasvedst* 147 humanmetpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi DNase1 + Valvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq K3LPvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais dhypvevmlk* 148DNase1L3 msrelapllllllsihsalamricsfnvrsfgeskqedknamdvivkvikrcdiilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflykeklvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsvkeidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwligdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeealdvsdhfpvefklqssraftnskksvtlrkktkskrs* 149 humanMglekslvrllllvlillvlgwvqpslgkesrakkfqrqhmdsdsspsssstyc pan-nqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmh creaticitdcrltngsrypncayrtspkerhiivacegspyvpvhfdatv* ribonu- clease 150huVK3LP + metpaqllfllllwlpdttgresaaqkfqrqhmdpdgssinsptycnqmmkrrd mrib1 +mI mtngsckpvntfvhepladvqavcsqenvtcknrksncyksssalhitdchlkg gG2A-nskypncdykttqyqkhiivacegnpyvpvhfdatvleprgltikpsppckcpa C + 2Spnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnhlttksfsrtpgk * 151 huVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNaseWT-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hIgGW-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp NLG-pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdg hDNase1-vevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt 105-114iskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdilkiaafniqtfgetkmsnativsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgsedvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqxayglsdqlaqa isdhypvevmlk**152 huVK3LP- metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrnhRNaseWT- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hIgGWT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp NLG-pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdg hDNase1-vevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt 114Fiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdilkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqa isdhypvevmlk* 153huVK3LP- metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrnhRNaseWT- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hIgGWT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp NLG-pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdg hDNAse1-vevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt WTiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdilkiaafniqtfgetkmsnatlvsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqa isdhypvevmlk* 154hVK3LP- metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq (WT)-vsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdava hIgG1WTeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 155 hVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq A114F-vsavdsyyyddgcepcgndtfnrepfivrffsrftevrefaivplhaapgdava hIgG1WTeidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 156 hVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)4-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlagais hIgGl WTdhypvevmlkdlsggggsggggsggggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 157 hVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1Galvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq 105R; A11vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 4F(G4s)5-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli hIgG1pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlagais WTdhypvevmlkdlsggggsggggsggggsggggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsls pgk* 158 hVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnativsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)5-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlagais 2-hIgG1dhypvevmlkdlsggggsggggsggggsggggsggggslepkssdkthtcppcp WTapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg k* 159 hVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli hIgG1 WTpdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqaisdhypvevmlkdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 160 hVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn (MT)-dsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp hIgG1WTpcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl spgk* 161 hVK3Lp-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn (WT)-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg (G4S)4lngggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp k-evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq hIgG1WTdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 162 hVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNasemtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn (WT)-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg (G4S)5-gggsggggsggggslepkssdkthtcppcpapellggpsvflfppkpkdtlmis 2lnk-rtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltv hIgG1WTlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 163 hVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNasemtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn (WT)-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp hIgG1 WTpcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl spgk* 164 murinemgsqtlphghmqtlifldleatglpssrpevtelcllavhrralentsisqghp Trex1ppvprpprvvdklslciapgkacspgaseitglskaelevqgrqrfddnlaill (FL)-raflqrqpqpcclvahngdrydfpllqtelarlstpspldgtfcvdsiaalkal trans-eqasspsgngsrksyslgsiytrlywqaptdshtaegdvltllsicqwkpqall criptqwvdeharpfstvkpmygtpattgttnlrphaatattplatangspsngrsrrp variantkspppekvpeapsqegllaplslltlltlaiatlyglflaspgq* 1 165 mousemgsqtlphghmqtlifldleatglpssrpevtelcllavhrralentsisqghp Trex1minppvprpprvvdklslciapgkacspgaseitglskaelevqgrqrfddnlaill ecraflqrqpqpcclvahngdrydfpllqtelarlstpspldgtfcvdsiaalkaleqasspsgngsrksyslgsiytrlywqaptdshtaegdvltllsicqwkpqallqwvdeharpfstvkpmygtpattgttdle 166 murinemetpaqllfllllwlpdttgmgsqtlphghmqtlifldleatglpssrpevtel Trex1-cllavhrralentsisqghpppvprpprvvdklslciapgkacspgaseitgls (G4S)4-kaelevqgrqrfddnlaillraflqrqpqpcclvahngdrydfpllqtelarls mIgG2a-ctpspldgtfcvdsiaalkaleqasspsgngsrksyslgsiytrlywqaptdshtaegdvltllsicqwkpqallqwvdeharpfstvkpmygtpattgttdlsggggsggggsggggsggggsleprgptikpsppckcpapnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnhlttksfsrtpgk* 167 murinemetpaqllfllllwlpdttgmgsqtlphghmqtlifldleatglpssrpevtel Trex1-cllavhrralentsisqghpppvprpprvvdklslciapgkacspgaseitgls (G4S)5-kaelevqgrqrfddnlaillraflqrqpqpcclvahngdrydfpllgtelarls mIgG2a-ctpspldgtfcvdsiaalkaleqasspsgngsrksyslgsiytrlywqaptdshtaegdvltllsicqwkpqallqwvdeharpfstvkpmygtpattgttdlsggggsggggsggggsggggsggggsleprgptikpsppckcpapnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnhlttksfsrtpgk* 168 NLGlnk vdgaaaspvnvsspsvqdi169 Murine metpaqllfllllwlpdttgmgsqtlphghmqtlifldleatglpssrpevtel Trex-cllavhrralentsisqghpppvprpprvvdklslciapgkacspgaseitgls Trex1-kaelevqgrqrfddnlaillraflqrqpqpcclvahngdrydfpllqtelarls (G4s)5-tpspldgtfcvdsiaalkaleqasspsgngsrksyslgsiytrlywqaptdsht mIgG2a-caegdvltllsicqwkpqallqwvdeharpfstvkpmygtpattgttdlmgsqtlphghmqtlifldleatglpssrpevtelcllavhrralentsisqghpppvprpprvvdklslciapgkacspgaseitglskaelevqgrqrfddnlaillraflqrqpqpcclvahngdrydfpllqtelarlstpspldgtfcvdsiaalkaleqasspsgngsrksyslgsiytrlywqaptdshtaegdvltllsicqwkpqallqwvdeharpfstvkpmygtpattgttdlsggggsggggsggggsggggsggggsleprgptikpsppckcpapnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnh lttksfsrtpgk* 170huVK3LP- metpaqllfllllwlpdttgmgpgarrqgrivqgrpemcfcppptplpplrilt huTREX1-lgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvte 72aa-lcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitgl (g4s)4-stavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllgaelaml IgG1 WTgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartkdlsggggsggggsggggsggggslepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 171 huVK3LP-metpaqllfllllwlpdttgmgpgarrqgrivqgrpemcfcppptplpplrilt huTREX1-lgthtptpcsspgsaagtyptmgsgalppgpmqtliffdmeatglpfsqpkvte 72aa-lcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitgl (g4s)5-stavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelaml hIgG1 WTgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlyggsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartkdlsggggsggggsggggsggggsggggslepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk* 172 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)4-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac hIgG1-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg WT-NLG-tacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgc hRNase1-gagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttc WTtcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 173 huVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)4-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlagais hIgG1-dhypvevmlkdlsggggsggggsggggsggggstglepkssdkthtcppcpape WT-NLG-llggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhna hRNase1-ktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakg WTqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst* 174 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNAseG8tggctcccagataccaccggtccttccctgggcaaggaatcccgggccaagaaa 8D-ttccagcggcagcatatggacccagacagttcccccagcagcagctccacctac hIgG1-tgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtg P238S; K3aacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaa 22S; P331aaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatg Scacatcacagactgccgcctgacaaacgactccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgtccagcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggacggcatggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtccccaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaacaccacgcctcccgtgctggactccgacggctccttctccctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaatgataatctaga 175 huVK3LP-metpaqllfllllwlpdttgpslgkesrakkfqrqhmdsdsspsssstycnqmm hRNAseG8rrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdc 8D-rltndsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkt hIgG1-htcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnw P238S; K3yvdgmevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkalpas 22S; P331iektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesng Sqpennynttppvldsdgsfslyskltvdksrwqqgnvfscsvmhealhnhytqk slslspgk* 176huVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)5-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac 1-hIgG1-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg WT-NLG-tacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgc hRNase1-gagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttc WTtcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 177 huVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)5-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais 1-hIgG1-dhypvevmlkdlsggggsggggsggggsggggsggggstglepkssdkthtcpp WT-NLG-cpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv hRNase1-evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekti WTskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst* 178 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)5-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac 2-hIgG1-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg WT-NLG-tacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgc hRNase1-gagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttc WTtcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtggctcaggtggtggaggatctggaggaggtgggagtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 179 huVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)5-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais 2-hIgG1-dhypvevmlkdlsggggsggggsggggsggggaggggslepkssdkthtcppcp WT-NLG-apellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvev hRNase1-hnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektisk WTakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst 180 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc hIgG1-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac WT-NLG-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg hRNasse1-tacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgc WTgagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 181 huVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1alvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdsyyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli hIgG1-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlagais WT-NLG-dhypvevmlkdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp hRNasse1-evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq WTdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivaceg spyvpvhfdasvedst*182 huVK3LP- gaccaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactmDNase1L ctggctcccagataccaccggtctaaggctctgctccttcaatgtgaggtcctt 3-tggagcgagcaagaaggaaaaccatgaagccatggatatcattgtgaagatcat mIgG2A-Ccaaacgctgtgaccttatactgttgatggaaatcaaggacagcagcaacaacat (mut)ctgtcccatgctgatggagaagctgaatggaaattcacgaagaagcacaacatacaactatgtgattagttctcgacttggaagaaacacgtacaaagagcagtatgccttcgtctacaaggagaagctggtgtctgtgaagacaaaataccactaccatgactatcaggatggagacacagacgtgttttccagggagccctttgtggtttggttccattccccctttactgctgtcaaggacttcgtgattgtccccttgcacacaactcccgagacctccgttaaagagatagatgagctggtcgatgtctacacggatgtgagaagccagtggaagacagagaatttcatcttcatgggtgatttcaacgccggctgtagctatgtccccaagaaggcctggcagaacattcgtttgaggacggaccccaagtttgtttggctgattggggaccaagaggacactacggtcaagaagagtaccagctgtgcctatgacaggattgtgctttgtggacaagagatagtcaactccgtggttccccgttccagtggcgtctttgactttcagaaagcttatgacttgtctgaagaggaggccctggatgtcagtgatcactttccagttgagtttaagctacagtcttcaagggccttcaccaacaacagaaaatctgtttctctcaaaaagagaaaaaaaggcaatcgctcctcagatctcgagcccagaggtctcacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatggtcacatgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctcggactccgggtaaatgataatctagaa 183 huVK3LP-metpaqllfllllwlpdttglrlcsfnvrsfgaskkenheamdiivkiikrcdl mDNase1Lillmeikdssnnicpmlmeklngnsrrsttynyvissrlgrntykeqyafvyke 3-klvsvktkyhyhdyqdgdtdvfsrepfvvwfhspftavkdfvivplhttpetsv mIgG2A-Ckeidelvdvytdvrsqwktenfifmgdfnagcsyvpkkawqnirlrtdpkfvwl (mut)igdqedttvkkstscaydrivlcgqeivnsvvprssgvfdfqkaydlseeealdvsdhfpvefklqssraftnnrksvslkkrkkgnrssdleprgltikpsppckcpapnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnhlttksfsrtpg k* 184 mDNase1Lgagaccagcttgccccatgtccctgcacccagcttccccacgcctggcctccct 3-NL-gctgctcttcatccttgccctccatgacaccctggccctaaggctctgctcctt mIgG2A_Ccaatgtgaggtcctttggagcgagcaagaaggaaaaccatgaagccatggatat (mut)cattgtgaagatcatcaaacgctgtgaccttatactgttgatggaaatcaaggacagcagcaacaacatctgtcccatgctgatggagaagctgaatggaaattcacgaagaagcacaacatacaactatgtgattagttctcgacttggaagaaacacgtacaaagagcagtatgccttcgtctacaaggagaagctggtgtctgtgaagacaaaataccactaccatgactatcaggatggagacacagacgtgttttccagggagccctttgtggtttggttccattccccctttactgctgtcaaggacttcgtgattgtccccttgcacacaactcccgagacctccgttaaagagatagatgagctggtcgatgtctacacggatgtgagaagccagtggaagacagagaatttcatcttcatgggtgatttcaacgccggctgtagctatgtccccaagaaggcctggcagaacattcgtttgaggacggaccccaagtttgtttggctgattggggaccaagaggacactacggtcaagaagagtaccagctgtgcctatgacaggattgtgctttgtggacaagagatagtcaactccgtggttccccgttccagtggcgtctttgactttcagaaagcttatgacttgtctgangaggangccctggatgtcagtgatcactttccagttgagtttaagctacagtcttcaagggccttcaccaacaacagaaaatctgtttctctcaaaaagagaaaaaaaggcaatcgctcctcagatctcgagcccagaggtctcacaatcaagccctctcctccatgcaaatgcccagcacctaacctcttgggtggatcatccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatggtcacatgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggactggatgagtggcaaggagttcaaatgctcggtcaacaacaaagacctcccagcgtccatcgagagaaccatctcaaaacccagagggccagtaagagctccacaggtatatgtcttgcctccaccagcagaagagatgactaagaaagagttcagtctgacctgcatgatcacaggcttcttacctgccgaaattgctgtggactggaccagcaatgggcgtacagagcaaaactacaagaacaccgcaacagtcctggactctgatggttcttacttcatgtacagcaagctcagagtacaaaagagcacttgggaaagaggaagtcttttcgcctgctcagtggtccacgagggtctgcacaatcaccttacgactaagagcttctctcggactccgggtaaatgataatctagaa 185 mDNase1Lmslhpasprlaslllfilalhdtlalrlcsfnvrsfgaskkenheamdiivkii 3-NL-krcdlillmeikdssnnicpmlmeklngnsrrsttynyvissrlgrntykeqya mIgG2A_Cfvykeklvsvktkyhyhdyqdgdtdvfsrepfvvwfhspftavkdfvivplhtt (mut)petsvkeidelvdvytdvrsqwktenfifmgdfnagcsyvpkkawqnirlrtdpkfvwligdqedttvkkstscaydrivlcgqeivnsvvprssgvfdfqkaydlsxexaldvsdhfpvefklqssraftnnrksvslkkrkkgnrssdleprgltikpsppckcpapnllggssvfifppkikdvlmislspmvtcvvvdvseddpdvqiswfvnnvevhtaqtqthredynstlrvvsalpiqhqdwmsgkefkcsvnnkdlpasiertiskprgpvrapqvyvlpppaeemtkkefsltcmitgflpaeiavdwtsngrteqnykntatvldsdgsyfmysklrvqkstwergslfacsvvheglhnhlttksf srtpgk* 186huVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1Ltggctcccagataccaccggtatgaggatctgctccttcaacgtcaggtccttt 3-ggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatc hIgG1Wt-aaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatc NLG-tgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatac hRNase1-aactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcc WTtttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgactatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 187 huVK3LP-metpaqllfllllwlpdttgmricsfnvrsfgeskqedknamdvivkvikrcdi hDNase1Lilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflyke 3-hIgG1-klvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsv WT-NLG-keidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwl hRNase1-igdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeeald WTvsdhfpvefklqssraftnskksvtlrkktkskrsdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst* 188 huVK3LP-aagcttgccgccatggaaaccccagcgcagcttctcttcctcctgctactctgg hRNase1-ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat WT-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg hIgG1-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac WT-NLG-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag hRNase1-aacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgc WTcgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 189 huVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp hIgG1-pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdg WT-NLG-vevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt hRNase1-iskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpen WTnykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst* 190 huVK3LP-aagcttgccgccatggaaaccccagcgcagcCtctcttcctcctgctactctgg hRNase1-ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat WT-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg (G4S)4-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac hIgG1-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag WT-NLG-aacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgc hRNase1-cgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaag WTgagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgCggaggtgcaCaatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcacgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 191 huVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg (G4S)4-gggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp hIgG1-evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq WT-NLG-dwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsl hRNase1-tclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqq WTgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivaceg spyvpvhfdasvedst*192 huVK3LP- aagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgghTREX1- ctcccagataccaccggtatgggccctggagctcgcagacagggcaggattgtg 72AA-cagggaaggcctgagatgtgcttctgcccaccccctaccccactccctcccctt (G4S)4-cggatcttaacactgggcactcacacacccaccccatgctcctctccaggctca hIgG1-gcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatg WT-NLG-cagaccctcatctttttcgacatggaggccactggcttgcccttctcccagccc hRNase1-aaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccc WTcccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagcccCgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataatctaga 193 huVK3LP-metpaqllfllllwlpdttgmgpgarrqgrivqgrpemcfcppptplpplrilt hTREX1-lgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvte 72AA-lcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitgl (G4S)4-stavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelaml hIgG1-gltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdsh WT-NLG-taegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartkdlsgggg hRNase1-sggggsggggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdt WTlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvpvhfdasvedst* 194 huVK3LP-aagcttgccgccatggaaaccccagcgcagcttctcttcctcctgctactctgg hRNase1-ctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcagcat WT-atggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatg hIgG1-aggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcac WT-NLG-gagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaag hTREX1-aacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgc 72AAcgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcatgggccctggagctcgcagacagggcaggattgtgcagggaaggcctgagatgtgcttctgcccaccccctaccccactccctccccttcggatcttaacactgggcactcacacacccaccccatgctcctctccaggctcagcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaatga taatctaga 195huVK3LP- metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlepkssdkthtcp hIgG1-pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdg WT-NLG-vevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiekt hTREX1-iskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpen 72AAnykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdimgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyptmgsgalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitglstavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartk* 196 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1Ltggctcccagataccaccggtatgaggatctgctccttcaacgtcaggtccttt 3-ggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatc (G4S)4-aaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatc hIgG1Wt-tgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatac NLG-aactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcc hRNase1-tttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgac WTtatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcccttccctgggcaaggaatcccgggccaagaaattccagcggcagcatatggactcagacagttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgcaagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagactgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccgaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacctaataa tctaga 197huVK3LP- metpaqllfllllwlpdttgmricsfnvrsfgeskqedknamdvivkvikrcdi hDNase1Lilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflyke 3-klvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsv (G4S)4-keidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwl hIgG1Wt-igdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeeald NLG-vsdhfpvefklqssraftnskksvtlrkktkskrsdlsggggsggggsggggsg hRNase1-gggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcv WTvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmheglhnhytqkslslspgkvdgasshvnvsspsvqdipslgkesrakkfqrqhmdsdsspsssstycnqmmrrrnmtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltngsrypncayrtspkerhiivacegspyvp vhfdasvedst* 198huVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNase1-tggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag WT-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg (G4S)4-atgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtg hIgG1-cacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgc WT-NLG-aagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagac hDNase1Ltgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccg 3aaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcatgaggatctgctccttcaacgtcaggtcctttggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatcaaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatctgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatacaactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcctttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgactatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcctaatgatctaga 199 huVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg (G4S)4-gggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp hIgG1-evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq WT-NLG-dwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsl hDNase1Ltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqq 3gnvfscsvmhealhnhytqkslslspgkvdgasshvnvsspsvqdimricsfnvrsfgeskqedknamdvivkvikrcdiilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflykeklvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsvkeidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwligdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeealdvsdhfpvefklqssraftnskksvtlrk ktkskrs* 200huVK3LP- gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1-tggctcccagataccaccggtctgaagatcgcagccttcaacatccagacattt G105R; A1ggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctg 14F-agccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgcc (G4S)4-gtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactac hIgG1-gtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtg WT-NLG-tacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgc hTREX1-gagccctgcaggaacgacaccttcaaccgagagccattcattgtcaggttcttc 72AAtcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggcttggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccaggctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcatgggccctggagctcgcagacagggcaggattgtgcagggaaggcctgagatgtgcttctgcccaccccctaccccactccctccccttcggatcttaacactgggcactcacacacccaccccatgctcctctccaggctcagcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaatgataatctaga 201 huVK3LP-metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi hDNase1-alvqevrdshlcavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdq G105R; A1vsavdayyyddgcepcrndtfnrepfivrffarftevrefaivplhaapgdava 14F-eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli (G4S)4-pdsadttatpthcaydrivvagmllrgavvpdsalpfnfqaayglsdqlaqais hIgG1-dhypvevmlkdlsggggsggggsggggsggggstglepkssdkthtcppcpape WT-NLG-llggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhna hTREX1-ktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakg 72AAqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscavmhealhnhytqkslslspgkvdgasshvnvsspsvqdimgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgppptvpppprvvdklslcvapgkacapaaseitglstavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartk* 202 huVK3LP-aagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactctgg hTREX1-ctcccagataccaccggtatgggccctggagctcgcagacagggcaggattgtg 72AAcagggaaggcctgagatgtgcttctgcccaccccctaccccactccctcccctt (G4S)4-cggatcttaacactgggcactcacacacccaccccatgctcctctccaggctca hIgG1-gcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatg WT-NLG-cagaccctcatctttttcgacatggaggccactggcttgcccttctcccagccc hDNase1-aaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccc G105R; A1cccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagac 14Faagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcctgaagatcgcagccttcaacatccagacatttggggagaccaagatgtccaatgccaccctcgtcagctacattgtgcagatcctgagccgctatgacatcgccctggtccaggaggtcagagacagccacctgactgccgtggggaagctgctggacaacctcaatcaggatgcaccagacacctatcactacgtggtcagtgagccactgggacggaacagctataaggagcgctacctgttcgtgtacaggcctgaccaggtgtctgcggtggacagctactactacgatgatggctgcgagccctgcgggaacgacaccttcaaccgagagccagccattgtcaggttcttctcccggttcacagaggtcagggagtttgccattgttcccctgcatgcggccccgggggacgcagtagccgagatcgacgctctctatgacgtctacctggatgtccaagagaaatggggctcggaggacgtcatgttgatgggcgacttcaatgcgggctgcagctatgtgagaccctcccagtggtcatccatccgcctgtggacaagccccaccttccagtggctgatccccgacagcgctgacaccacagctacacccacgcactgtgcctatgacaggatcgtggttgcagggatgctgctccgaggcgccgttgttcccgactcggctcttccctttaacttccagnctgcctatggcctgagtgaccaactggcccaagccatcagtgaccactatccagtggaggtgatgctgaagtgataatctaga 203 huVK3LP-metpaqllfllllwlpdttgmgpgarrqgrivqgrpemcfcppptplpplrilt hTREX1-lgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvte 72AAlcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitgl (G4S)4-stavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelaml hIgG1-gltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdsh WT-NLG-taegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartkdlsgggg hDNase1-sggggsggggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdt G105R; A1lmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvs 14Fvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasshvnvsspsvqdilkiaafniqtfgetkmsnativsyivqilsrydialvqevrdshltavgklldnlnqdapdtyhyvvseplgrnsykerylfvyrpdqvsavdsyyyddgcepcgndtfnrepaivrffsrftevrefaivplhaapgdavaeidalydvyldvqekwgsedvmlmgdfnagcsyvrpsqwssirlwtsptfqwlipdsadttatpthcaydrivvagmllrgavvpdsalpfnfqxayglsdqlaqaisdhypvevmlk* 204 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hRNase1-tggctcccagataccaccggtaaggaatcccgggccaagaaattccagcggcag WT-catatggactcagacagttcccccagcagcagctccacctactgtaaccaaatg (G4S)4-atgaggcgccggaatatgacacaggggcggtgcaaaccagtgaacacctttgtg hIgG1-cacgagcccctggtagatgtccagaatgtctgtttccaggaaaaggtcacctgc WT-NLG-aagaacgggcagggcaactgctacaagagcaactccagcatgcacatcacagac hTREX1-tgccgcctgacaaacggctccaggtaccccaactgtgcataccggaccagcccg 72AAaaggagagacacatcattgtggcctgtgaagggagcccatatgtgccagtccactttgatgcttctgtggaggactctacagatctctccggaggaggtggctcaggtggtggaggatctggaggaggtgggagtggtggaggtggttctaccggtctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggtgctagcagccatgtgaatgtgagcagccctagcgtgcaggatatcatgggccctggagctcgcagacagggcaggattgtgcagggaaggcctgagatgtgcttctgcccaccccctaccccactccctccccttcggatcttaacactgggcactcacacacccaccccatgctcctctccaggctcagcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaatgataatctaga 205 huVK3LP-metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspsssstycnqmmrrrn hRNase1-mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn WT-gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg (G4S)4-gggsggggstglepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtp hIgG1-evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq WT-NLG-dwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsl hTREX1-tclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqq 72AAgnvfscsvmhealhnhytqkslslspgkvdgasshvnvsspsvqdimgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitglstavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartk* 206 huVK3LP-gttaagcttgccaccatggaaaccccagcgcagcttctcttcctcctgctactc hDNase1Ltggctcccagataccaccggtatgaggatctgctccttcaacgtcaggtccttt 3-hIgG1-ggggaaagcaagcaggaagacaagaatgccatggatgtcattgtgaaggtcatc WT-NLG-aaacgctgtgacatcatactcgtgatggaaatcaaggacagcaacaacaggatc hTREX1-tgccccatactgatggagaagctgaacagaaattcaaggagaggcataacatac 72AAaactatgtgattagctctcggcttggaagaaacacatataaagaacaatatgcctttctctacaaggaaaagctggtgtctgtgaagaggagttatcactaccatgactatcaggatggagacgcagatgtgttttccagggagccctttgtggtctggttccaatctccccacactgctgtcaaagacttcgtgattatccccctgcacaccaccccagagacatccgttaaggagatcgatgagttggttgaggtctacacggacgtgaaacaccgctggaaggcggagaatttcattttcatgggtgacttcaatgccggctgcagctacgtccccaagaaggcctggaagaacatccgcttgaggactgaccccaggtttgtttggctgatcggggaccaagaggacaccacggtgaagaagagcaccaactgtgcatatgacaggattgtgcttagaggacaagaaatcgtcagttctgttgttcccaagtcaaacagtgtttttgacttccagaaagcttacaagctgactgaagaggaggccctggatgtcagcgaccactttccagttgaatttaaactacagtcttcaagggccttcaccaacagcaaaaaatctgtcactctaaggaagaaaacaaagagcaaacgctcagatctcgagcccaaatcttctgacaaaactcacacatgtccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctctctgtctccgggtaaagtcgacggagctagcagccccgtgaacgtgagcagccccagcgtgcaggatatcatgggccctggagctcgcagacagggcaggattgtgcagggaaggcctgagatgtgcttctgcccaccccctaccccactccctccccttcggatcttaacactgggcactcacacacccaccccatgctcctctccaggctcagcagcaggtacgtacccaaccatgggctcgcaggccctgcccccggggcccatgcagaccctcatctttttcgacatggaggccactggcttgcccttctcccagcccaaggtcacggagctgtgcctgctggctgtccacagatgtgccctggagagcccccccacctctcaggggccacctcccacagttcctccaccaccgcgtgtggtagacaagctctccctgtgtgtggctccggggaaggcctgcagccctgcagccagcgagatcacaggtctgagcacagctgtgctggcagcgcatgggcgtcaatgttttgatgacaacctggccaacctgctcctagccttcctgcggcgccagccacagccctggtgcctggtggcacacaatggtgaccgctacgacttccccctgctccaagcagagctggctatgctgggcctcaccagtgctctggatggtgccttctgtgtggatagcatcactgcgctgaaggccctggagcgagcaagcagcccctcagaacacggcccaaggaagagctacagcctaggcagcatctacactcgcctgtatgggcagtcccctccagactcgcacacggctgagggtgatgtcctggccctgctcagcatctgtcagtggagaccacaggccctgctgcggtgggtggatgctcacgccaggcctttcggcaccatcaggcccatgtatggggtcacagcctctgctaggaccaaatgataatctaga 207 huVK3LP-metpaqllfllllwlpdttgmricsfnvrsfgeskqedknamdvivkvikrcdi hDNase1Lilvmeikdsnnricpilmeklnrnsrrgitynyvissrlgrntykeqyaflyke 3-hIgG1-klvsvkrsyhyhdyqdgdadvfsrepfvvwfqsphtavkdfviiplhttpetsv WT-NLG-keidelvevytdvkhrwkaenfifmgdfnagcsyvpkkawknirlrtdprfvwl hTREX1-igdqedttvkkstncaydrivlrgqeivssvvpksnsvfdfqkayklteeeald 72AAvsdhfpvefklqssraftnskksvtlrkktkskrsdlepkssdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkvdgasspvnvsspsvqdimgpgarrqgrivqgrpemcfcppptplpplriltlgthtptpcsspgsaagtyptmgsqalppgpmqtliffdmeatglpfsqpkvtelcllavhrcalespptsqgppptvpppprvvdklslcvapgkacspaaseitglstavlaahgrqcfddnlanlllaflrrqpqpwclvahngdrydfpllqaelamlgltsaldgafcvdsitalkalerasspsehgprksyslgsiytrlygqsppdshtaegdvlallsicqwrpqallrwvdaharpfgtirpmygvtasartk*

1. A hybrid nuclease molecule comprising a first nuclease domain and amodified Fc domain, wherein the first nuclease domain is operativelycoupled to the Fc domain. 2.-63. (canceled)
 64. The hybrid nucleasemolecule of claim 1 wherein the Fc domain is modified to decreasebinding to Fcγ receptors or complement proteins or both.
 65. The hybridnuclease molecule of claim 64 which has a reduced effector functionselected from the group consisting of opsonization, phagocytosis,complement dependent cytotoxicity, and antibody-dependent cellularcytotoxicity.
 66. The hybrid nuclease molecule of claim 1, furthercomprising a second nuclease domain operatively coupled to the Fcdomain.
 67. The hybrid nuclease molecule of claim 1, wherein the firstnuclease domain is operatively coupled to the Fc domain via a firstlinker domain
 68. The hybrid nuclease molecule of claim 67, wherein thefirst linker domain is a polypeptide linker, such as a gly-ser linker.69. The hybrid nuclease molecule of claim 66, wherein the secondnuclease domain is operatively coupled to the Fc domain via a secondlinker domain.
 70. The hybrid nuclease molecule of claim 69, wherein thesecond linker domain is a polypeptide linker, such as an NLG peptide.71. The hybrid nuclease molecule of claim 1, wherein the first nucleasedomain comprises an RNase.
 72. The hybrid nuclease molecule of claim 71,wherein the RNase is a human RNase, such as a human pancreatic RNase A.73. The hybrid nuclease molecule of claim 1, wherein the Fc domaincomprises a human immunoglobulin Fc domain, such as a human IgG1 Fcdomain.
 74. The hybrid nuclease molecule of claim 73, wherein the Fcdomain comprises a hinge domain, a CH2 domain and a CH3 domain.
 75. Thehybrid nuclease molecule of claim 66, wherein the second nuclease domaincomprises a DNase.
 76. The hybrid nuclease molecule of claim 75, whereinthe DNase is selected from the group consisting of a Type I human DNase,a human DNase 1L3, or human TREX1.
 77. The hybrid nuclease molecule ofclaim 1 comprising a polypeptide, wherein the first nuclease domaincomprises an amino acid sequence of a mature peptide of a human RNasepancreatic precursor set forth in SEQ ID NO:149, or an RNase comprisingan amino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:149.
 78. The hybrid nuclease molecule of claim 1,wherein the Fc domain is a human IgG1 Fc domain comprising an amino acidsequence set forth in SEQ ID NO:145, or an Fc domain comprising an aminoacid sequence at least 90% identical to the amino acid sequence setforth in SEQ ID NO:145.
 79. The hybrid nuclease molecule of claim 66comprising a polypeptide, wherein the second nuclease domain comprises ahuman DNase comprising an amino acid sequence set forth in SEQ IDNO:143, or a DNase comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:143.
 80. Thehybrid nuclease molecule of claim 1, wherein the Fc domain comprises amodified hinge domain comprising at least one substitution.
 81. Thehybrid nuclease molecule of claim 80, wherein the modified hinge domaincomprises at least one cysteine substitution, such as a substitutionwith serine.
 82. The hybrid nuclease molecule of claim 1, wherein the Fcdomain comprises a modified CH2 domain comprising at least onesubstitution.
 83. The hybrid nuclease molecule of claim 82, wherein themodified CH2 domain comprises at least one substitution of an amino acidassociated with Fcγ receptor binding or complement protein binding orboth.
 84. The hybrid nuclease molecule of claim 83, wherein thesubstitution is selected from the group consisting of P238S, P331S,N297S or combination thereof.
 85. A hybrid nuclease molecule comprisinga polypeptide, wherein the polypeptide comprises an amino acid sequenceof a mature peptide of a human RNase pancreatic precursor operativelylinked to an Fc domain as set forth in SEQ ID NO:163, or a polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:163.
 86. A hybrid nuclease moleculecomprising a polypeptide, wherein the polypeptide comprises an aminoacid sequence of a mature peptide of a human RNase pancreatic precursoroperatively linked to an Fc domain via a linker as set forth in SEQ IDNO:161, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:161.
 87. Ahybrid nuclease molecule comprising a polypeptide, wherein thepolypeptide comprises an amino acid sequence of a mature peptide of ahuman RNase pancreatic precursor operatively linked to the N-terminus ofan Fc domain and a human DNase amino acid sequence operatively linked tothe C-terminus of the Fc domain as set forth in SEQ ID NO:153, or apolypeptide comprising an amino acid sequence at least 90% identical tothe amino acid sequence set forth in SEQ ID NO:153.
 88. The hybridnuclease molecule of claim 86, wherein the Fc domain comprises amodified CH2 domain comprising at least one substitution selected fromthe group consisting of P238S, P331S, N297S or combination thereof. 89.A composition comprising the hybrid nuclease molecule of claim 1 and apharmaceutically acceptable carrier.
 90. A nucleic acid moleculeencoding the hybrid nuclease molecule according to claim
 1. 91. Arecombinant expression vector comprising a nucleic acid moleculeaccording to claim
 90. 92. A host cell transformed with the recombinantexpression vector according to claim
 91. 93. A method of making thehybrid nuclease molecule of claim 1, comprising: providing a host cellcomprising a nucleic acid sequence that encodes the hybrid nucleasemolecule; and maintaining the host cell under conditions in which thehybrid nuclease molecule is expressed.
 94. A method for treating orpreventing a condition associated with an abnormal immune response,comprising administering to a subject an effective amount of a hybridnuclease molecule of claim
 1. 95. The method of claim 94, wherein thecondition is an autoimmune disease.
 96. The method of claim 95, whereinthe autoimmune disease is selected from the group consisting ofinsulin-dependent diabetes mellitus, multiple sclerosis, experimentalautoimmune encephalomyelitis, rheumatoid arthritis, experimentalautoimmune arthritis, myasthenia gravis, thyroiditis, an experimentalform of uveoretinitis, Hashimoto's thyroiditis, primary myxoedema,thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis,Addison's disease, premature menopause, male infertility, juvenilediabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid,sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolyticanaemia, idiopathic leucopenia, primary biliary cirrhosis, activechronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis,Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis,dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), andconnective tissue disease.
 97. The method of claim 96, wherein theautoimmune disease is SLE.
 98. A method of treating SLE comprisingadministering to a subject a nuclease-containing composition in anamount effective to digest immune complexes containing RNA, DNA or bothRNA and DNA, wherein the composition comprises a pharmaceuticallyacceptable carrier and an isolated polypeptide comprising a firstnuclease domain.
 99. The method of claim 98, wherein the first nucleasedomain comprises an RNase.
 100. The method of claim 98, wherein thepolypeptide further comprises an Fc domain operatively coupled to thefirst nuclease domain.
 101. The method of claim 100, wherein the Fcdomain is modified to decrease binding to Fcγ receptors or complementproteins or both.
 102. The method of claim 101, wherein the Fc domainhas a reduced effector function selected from the group consisting ofopsonization, phagocytosis, complement dependent cytotoxicity, andantibody-dependent cellular cytotoxicity.
 103. The method of claim 98,wherein the polypeptide further comprises a second nuclease domaincomprising a DNase.